Stabilizing alkylglycoside compositions and methods thereof

ABSTRACT

The present invention relates to alkylglycoside-containing compositions and methods for increasing the stability, reducing the aggregation and immunogenicity, increasing the biological activity, and reducing or preventing fibrillar formation of a peptide, polypeptide, or variant thereof, for example insulin and Peptide T or analog thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/474,055 filed Jun. 23, 2006, pending. The disclosure of the priorapplication is considered part of and is incorporated by reference inthe disclosure of this application.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methodsthereof that increase stability, reduce aggregation and immunogenicity,increase biological activity, and reduce or prevent fibrillar formationof peptides or proteins in therapeutically useful formulations, andspecifically, to compositions having at least one peptide or proteindrug and at least one alkylglycoside or saccharide alkyl estersurfactant.

BACKGROUND INFORMATION

Proteins undergo numerous physical and chemical changes that affectpotency and safety. Among these are aggregation, which includesdimerization, trimerization, and higher-order aggregates, pluscrystallization and precipitation. Aggregation is rapidly emerging as akey issue underlying multiple deleterious effects for peptide orprotein-based therapeutics, including loss of efficacy, alteredpharmacokinetics, reduced stability or product shelf life, and inductionof unwanted immunogenicity. In addition, bioavailability andpharmacokinetics of a self-associating peptide can be influenced byaggregate size and the ease of disruption of the non-covalentintermolecular interactions at the subcutaneous site. Hydrophobicaggregation mediated by seemingly innocuous solution formulationconditions can have a dramatic effect on the subcutaneousbioavailability and pharmacokinetics of a therapeutic peptide and in theextreme, can totally preclude its absorption (Clodfelter 1998). Duringthe course of the manufacturing process, proteins are purified andconcentrated using a variety of means. These means includeultrafiltration, affinity chromatography, selective absorptionchromatography, ion exchange chromatography, lyophilization, dialysis,and precipitation or salting-out. Such concentration can lead toaggregation which in turn can increase the immunogenicity of the proteintherapeutic. One means to avoid this problem is to work with the proteinsolutions at lower concentrations and correspondingly larger volumes.However, the need to work with larger volumes naturally introducesinefficiencies in the manufacturing process. During fill-and-finishoperations, concentrated protein solutions squeeze through piston pumps,which imparts high-shear and mechanical stresses that cause denaturationand aggregation. By adding alkylglycosides as described in the presentinvention to the protein solutions during the course of purification andconcentration by the means described above, aggregation can be reducedor eliminated, providing for greater efficiency in the manufacturingprocess, and providing for a final product which is desirably lessimmunogenic. The concentrations of alkylglycoside found to be effectivein this application must be at least somewhat higher than the criticalmicelle concentration.

Many products are only effective when delivered by injection inrelatively high concentration. Preventing aggregation has become a majorissue for pharmaceutical formulators since the trend towardhigh-concentration solutions increases the likelihood of protein-proteininteractions favoring aggregation. Thus, protein aggregation may impactbiological product process yield and potency. Since aggregation isfrequently mediated by higher temperatures, protein therapeutics requirecertain so-called “Cold Chain” handling requirements to guarantee acontinuous chain of refrigerated temperatures during shipping andstorage (DePalma Jan. 15, 2006). Cold chain requirements significantlyincrease the cost of storing and transporting drugs. The presentinvention mitigates and, in some cases, may eliminate the need forstrict cold-chain maintenance.

Over the last five years, the FDA and other regulatory agencies haveincreased their scrutiny of aggregation events, and thusbiopharmaceutical companies have increased their efforts to understandthem. Of particular concern is the induction of unwanted immunogenicity.The immunogenicity of a self-associating peptide can be influenced bythe formation of aggregates formed as a result of non-covalentintermolecular interactions. For example, interferon has been shown toaggregate resulting in an antibody response (Hermeling et al. 2006). Theantibody response to erythropoietin has been shown to produce “pure redcell aplasia” in a number of patients receiving recombinant EPO,(Casadevall et al. 2002) which is potentially a life threatening sideeffect of EPO therapy. Insulin is well known to lose activity rapidly asa result of protein aggregation upon agitation at temperatures abovethose found upon refrigerated storage (Pezron et al. 2002; Sluzky et al.1991). Aggregation of recombinant AAV2 results in reduced yield duringpurification and has deleterious effects on immunogenicity following invivo administration (Wright 2005). Monoclonal antibody basedtherapeutics have also been shown to be subject to inactivation as aresult of protein aggregation (King et al. 2002). The number ofmonoclonal antibodies in human clinical trials has been on the rise.Often monoclonal antibodies require relatively high dosing (in the 1 to2 mg/kg range) whether administered in a hospital setting by i.v.administration or in an outpatient setting in a clinic or at home by amore convenient mode of delivery such as subcutaneous administration.Development of antibody formulations at high concentrations posestability, manufacturing, and delivery challenges related to thepropensity of antibodies to aggregate at the higher concentrations.

Recombinant human factor VIII (rFVIII), a multidomain glycoprotein isused in replacement therapy for treatment of hemophilia A.Unfortunately, 15%-30% of the treated patients develop inhibitoryantibodies. The presence of aggregated protein in formulations isgenerally believed to enhance the antibody development response (Purohitet al. 2006).

Enzymes too are known to lose activity as a result of aggregation. Forexample thermal inactivation of urokinase occurs via aggregation (Porteret al. 1993).

In addition, hydrophobic aggregation mediated by seemingly innocuoussolution formulation conditions can have a dramatic effect on thesubcutaneous bioavailability and pharmacokinetics of a therapeuticpeptide and in the extreme, can totally preclude its absorption(Clodfelter et al. 1998). Peptide or protein therapeutics are frequentlyformulated at high concentration so that the volume of the formulationthat must be administered in order to achieve a therapeuticallyeffective dose can be kept small thereby minimizing patient discomfort.Unfortunately, high protein or peptide concentrations often induceaggregation. In addition, protein aggregation can be induced bynecessary excipients such as the antimicrobial preservative benzylalcohol which are included to maintain product sterility (Roy et al.2005).

Protein stabilization during lyophilization has also posed problems.Protein therapeutics frequently lose biological activity afterlyophilization and reconstitution as a result of aggregate formation andprecipitation. Several reconstitution medium additives have been foundto result in a significant reduction of aggregation. These includesulfated polysaccharides, polyphosphates, amino acids and varioussurfactants, not including alkylglycosides (Zhang et al. 1995). In somecases, a combination of alcohols, organic solvents, such as in Fortical,Unigene's nasally delivered calcitonin product, may be used. Roccatanoet al. (2002) have used trifluoroethanol mixtures to stabilize variouspolypeptides. Unfortunately, such agents may be harsh on mucosal tissuecausing patient discomfort or local toxicity.

SUMMARY OF THE INVENTION

The present invention relates generally to compositions that stabilize,reduce aggregation and immunogenicity of peptides or proteins intherapeutically useful formulations. More specifically, the presentinvention provides therapeutic compositions comprising at least oneself-associating, or self-aggregating, peptide or protein drug and atleast one surfactant, wherein the surfactant is further comprised of atleast one alkylglycoside and/or saccharide alkyl ester. Further, thepresent invention provides for compositions that when administered tovertebrates preclude or reduce aggregation thereby increasing theshelf-life of the therapeutic or increasing the range of conditions suchas temperature and agitation that may be tolerated without causing harmto the functional properties of the therapeutic.

Accordingly, in one aspect of the invention, there is provided apharmaceutical composition for increasing the stability, reducingaggregation or reducing immunogenicity of a therapeutically activepeptide, polypeptide or variant thereof consisting of a therapeuticallyactive peptide or polypeptide and variant thereof, and a stabilizingagent, wherein the stabilizing agent is at least one alkylglycoside, andwherein the alkylglycoside stabilizes the therapeutically activepeptide, polypeptide or variant thereof. The peptide, polypeptide orvariant thereof includes but is not limited to insulin or an analogthereof, interferon, a monoclonal antibody, erythropoietin, Peptide T oran analog thereof, D-alanine Peptide T amide (DAPTA), growth hormone,parathyroid hormone or active fragments PTH 1-34 or PTH 3-34, insulinand Hematide™. Also, the alkylglycoside of the invention includes but isnot limited dodecyl maltoside, tridecyl maltoside, tetradecyl maltoside,sucrose mono-dodecanoate, sucrose mono-tridecanoate, and sucrosemono-tetradecanoate.

In another aspect of the invention, there is provided a method forincreasing the stability of a therapeutically active peptide,polypeptide or variant thereof by admixing a therapeutically activepeptide, polypeptide or variant thereof, a stabilizing agent and abuffering agent, wherein the stabilizing agent is at least onealkylglycoside surfactant, wherein the surfactant increases thestability of the therapeutically active peptide, polypeptide or variantthereof.

The invention also provides for a method for reducing aggregation of atherapeutically active peptide, polypeptide or variant thereof byadmixing a therapeutically active peptide, polypeptide or variantthereof, an aggregation reducing agent, wherein the stabilizing agent isat least one alkylglycoside surfactant, wherein the surfactant reducesaggregation of the therapeutically active peptide, polypeptide orvariant thereof.

In yet another aspect of the invention, there is provided a method forreducing immunogenicity of a therapeutically active peptide, polypeptideor variant thereof upon administration to a vertebrate, by admixing atherapeutically active peptide, polypeptide or variant thereof, animmunogenicity reducing agent, wherein the immunogenicity reducing agentis at least one alkylglycoside or surfactant, wherein the surfactantreduces immunogenicity of the therapeutically active peptide,polypeptide or variant thereof.

In one aspect of the invention, there is a formulation for treating asubject having or at risk of having HIV, the formulation containing aprophylactically or therapeutically effective amount of a compositioncomprising D-alanine Peptide T amide (DAPTA), and at least onealkylglycoside to the subject.

In another aspect of the invention, there is an intranasal formulationfor treating a subject having or at risk of having HIV, the intranasalformulation containing a prophylactically or therapeutically effectiveamount of a composition comprising D-alanine Peptide T amide (DAPTA),and at least one alkylglycoside to the subject

Still, the invention provides a formulation for treating a subjecthaving or at risk of having a CCR5-mediated disease, the formulationcontaining a prophylactically or therapeutically effective amount of acomposition comprising D-alanine Peptide T amide (DAPTA) and at leastone alkylglycoside.

Still, the invention provides an intranasal formulation for treating asubject having or at risk of having a CCR5-mediated disease, theintranasal formulation containing a prophylactically or therapeuticallyeffective amount of a composition comprising D-alanine Peptide T amide(DAPTA) and at least one alkylglycoside.

In yet another aspect of the invention, there is provided a method oftreating a subject having or at risk of having HIV by administering aprophylactically or therapeutically effective amount of a compositioncomprising D-alanine Peptide T amide (DAPTA) and at least onealkylglycoside surfactant to the subject, thereby treating the subject.

The present invention also provides a method for treating aninflammatory disease by administering to a subject in need thereof atherapeutically effective amount of a therapeutically active peptide,polypeptide or variant composition containing a therapeutically activepeptide or polypeptide or variant thereof, a stabilizing agent, and abuffering agent, wherein the stabilizing agent is at least onealkylglycoside, wherein the therapeutically active peptide, polypeptideor variant thereof is a Peptide T or analog thereof.

Another aspect of the invention is a method of manufacturingnon-aggregated aqueous solutions of otherwise self-aggregatingtherapeutically active peptide, polypeptide or variant thereof byadmixing at least one alkylglycoside surfactant in an aqueous solutionof the self-aggregating therapeutically active peptide, polypeptide orvariant thereof and concentrating the therapeutically active peptide,polypeptide or variant thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing ordered fibrillar peptide aggregates packedin narrow parallel arrays of β sheets and stacked perpendicular to thelong axis of the fibril.

FIG. 2 is a graph showing light scatter readings for the polypeptideinsulin at pH 6.5, admixed with “A”, mono-dodecanoate (SDD) or “B”dodecyl maltoside (DDM).

FIG. 3 is a graph showing light scatter readings for the polypeptideinsulin at pH 7.4, admixed with “A”, mono-dodecanoate (SDD) or “B”dodecyl maltoside (DDM).

FIG. 4 is a graph showing light scatter readings for the polypeptidehuman growth hormone (hGH) at pH 6.5, admixed with either 0.124% or0.125% dodecyl maltoside (DDM).

FIG. 5 is a graph showing the time dependent effect of untreated DAPTAaggregation stored for different periods of time at 4 degrees Celcius (6h ♦) or 25 degrees Celcius (1▪, 3▴ and 4* weeks and 2● months).

FIG. 6 is a graph showing DAPTA admixed with TFE and/or dodecylmaltoside (“A3”), or sucrose mono dodecanoate (“B3”) inhibiting HIVinfection in macrophages.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of specific embodiments and the Examplesincluded therein.

The present invention describes formulations comprising at least onepeptide or protein, whether at high or low concentration, and at leastone alkylglycoside and/or saccharide alkyl ester surfactant, hereinaftertermed “alkylglycosides”. As used herein, “alkylglycoside” refers to anysugar joined by a linkage to any hydrophobic alkyl, as is known in theart. The linkage between the hydrophobic alkyl chain and the hydrophilicsaccharide can include, among other possibilities, a glycosidic, ester,thioglycosidic, thioester, ether, amide or ureide bond or linkage.Examples of which are described herein. The terms alkylglycoside andalkylsaccharide may be used interchangeably herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

The term, “stabilizing agent” or “stabilizer” as used herein is achemical or compound that is added to a solution or mixture orsuspension or composition or therapeutic composition to maintain it in astable or unchanging state; or is one which is used because it producesa reaction involving changes in atoms or molecules leading to a morestable or unchanging state.

The term “aggregate” or “aggregation” as used herein is means to cometogether or collect in a mass or whole, e.g., as in the aggregation ofpeptides, polypeptides, or variants thereof. Aggregates can beself-aggregating or aggregate due to other factors, e.g., aggregatingagents or precipitating agents, or antibodies, or other means andmethods whereby peptides, polypeptides, or variants thereof cause tocome together.

The term, “immunogenicity” as used herein is the degree to which asubstance induces an immune response; whereas, the term “antigenicity”is used to describe the capacity to induce an immune response.

The term “impart,” including grammatical variations thereof, as usedherein means to give or convey.

The term “promote,” including grammatical variations thereof, as usedherein means to help bring about.

The term “resistance,” including grammatical variations thereof, as usedherein means to retard or oppose a particular effect (e.g., opposeattachment of plasma factors which foul tissue interfacing devices).

The term “sterilize,” including grammatical variations thereof, as usedherein means to make substantially free of viable microbes.

As used herein, “drug” is any therapeutic compound or molecule includingbut not limited to nucleic acids, small molecules, polypeptide orpeptide, etc., The peptide may be any medically or diagnostically usefulpeptide or protein of small to medium size (i.e. up to about 75 kDa).The mechanisms of improved polypeptide absorption are described in U.S.Pat. No. 5,661,130 to Meezan et al., the reference of which is herebyincorporated in its entirety. The present invention can be mixed withall such peptides, although the degree to which the peptides benefitsare improved may vary according to the molecular weight and the physicaland chemical properties of the peptide, and the particular surfactantused. Examples of polypeptides include insulin like growth factor-I(IGF-I or Somatomedin-C), insulin, calcitonin, leptin, hGH, humanparathyroid hormone (PTH), melatonin, GLP-1 or Glucagon-like peptide-1,GiP, OB-3 peptide, pituitary adenylate cyclase neuropeptide—activatingpolypeptide (PACAP), GM-1 ganglioside, nerve growth factor (NGF),D-tryp6)-LHRH, nafarelin, FGF, VEGF, VEGF antagonists, Leuprolide,interferon-alpha, interferon-beta, interferon-gamma, low molecularweight heparin, PYY, LHRH, LH, GDNF, G-CSF, Ghrelin antagonists,Ghrelin, KGF, Imitrex, Integrelin, Nesiritide, Sandostatin, PTH (1-34),cetrorelix acetate, ganirelix acetate, bivalirudin, zafirlukast,Exanitide, pramlintide acetate, vasopressin, desmopressin, glucagon,ACTH, GHRH and analogs, oxytocin, corticotropin releasing hormone,TRHrh, atrial natriuretic peptide, thyroxine releasing hormone, FSH,prolactin, Tobramycin, Triptorelin, Goserelin, Fuzeon, Hematide,Buserelin, Octreotide, Gonadorelin, Felypressin, Deslorelin,Vasopressin, 8-L-Arg, Eptifibatide, GM-CSF, EPO, Interleukin-11,Endostatin, Angiostatin, N-acetyl oxyntomodulin 30-37, Oxyntomodulin,Ularitide, Xerecept, Apo A-IV, rNAPc2, Secretin, Thymopentin, NeuromedinU, Neurotensin, Thrombospondin-1 inhibitors, FGF-18, FGF-20, FGF-21,Elcatonin Acetate, Antide Acetate, Dynorphin A (1-13) Acetate,Sincalide, Thymopentin Acetate, Thymosin alpha1 acetate (Thymalfasin),Fertirelin Acetate, CRF Acetate, CRF (ovine), Hisrelin, Thymalfasin,Ecallantide, Oxycortin, Urocortin, Arixtra, Spiegelmer nucleotideaptamers, CGRP (calcitonin gene related protein), Urocortin, Amylin,IL-21, melanotan, valpreotide, and ACV-1 neuropathic pain peptide. Also,see Table I.

As used herein, a “therapeutic composition” can comprise an admixturewith an aqueous or organic carrier or excipient, and can be compounded,for example, with the usual non toxic, pharmaceutically acceptablecarriers for tablets, pellets, capsules, lyophilizates, suppositories,solutions, emulsions, suspensions, or other form suitable for use. Thecarriers, in addition to those disclosed above, can include alginate,collagen, glucose, lactose, mannose, gum acacia, gelatin, mannitol,starch paste, magnesium trisilicate, talc, corn starch, keratin,colloidal silica, potato starch, urea, medium chain lengthtriglycerides, dextrans, and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition, auxiliary stabilizing, thickening or coloring agents can beused, for example a stabilizing dry agent such as triulose.

As used herein, the term “therapeutic targets” may thus be defined asthose analytes which are capable of exerting a modulating force, wherein“modulation” is defined as an alteration in function inclusive ofactivity, synthesis, production, and circulating levels. Thus,modulation effects the level or physiological activity of at least oneparticular disease related biopolymer marker or any compound orbiomolecule whose presence, level or activity is linked either directlyor indirectly, to an alteration of the presence, level, activity orgeneric function of the biopolymer marker, and may includepharmaceutical agents, biomolecules that bind to the biopolymer markers,or biomolecules or complexes to which the biopolymer markers bind. Thebinding of the biopolymer markers and the therapeutic moiety may resultin activation (agonist), inhibition (antagonist), or an increase ordecrease in activity or production (modulator) of the biopolymer markersor the bound moiety. Examples of such therapeutic moieties include, butare not limited to, antibodies, oligonucleotides, proteins (e.g.,receptors), RNA, DNA, enzymes, peptides or small molecules. With regardto immunotherapeutic moieties, such a moiety may be defined as aneffective analog for a major epitope peptide which has the ability toreduce the pathogenicity of key lymphocytes which are specific for thenative epitope. An analog is defined as having structural similarity butnot identity in peptide sequencing able to be recognized by T-cellsspontaneously arising and targeting the endogenous self epitope. Acritical function of this analog is an altered T-cell activation whichleads to T-cell anergy or death.

As used herein, a “pharmaceutically acceptable carrier” or “therapeuticeffective carrier” is aqueous or non aqueous (solid), for examplealcoholic or oleaginous, or a mixture thereof, and can contain asurfactant, emollient, lubricant, stabilizer, dye, perfume,preservative, acid or base for adjustment of pH, a solvent, emulsifier,gelling agent, moisturizer, stabilizer, wetting agent, time releaseagent, humectant, or other component commonly included in a particularform of pharmaceutical composition. Pharmaceutically acceptable carriersare well known in the art and include, for example, aqueous solutionssuch as water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, and oils such as olive oil orinjectable organic esters. A pharmaceutically acceptable carrier cancontain physiologically acceptable compounds that act, for example, tostabilize or to increase the absorption of specific inhibitor, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients.

The pharmaceutical compositions can also contain other pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such “substances” include, but are not limited to, pHadjusting and buffering agents, tonicity adjusting agents and the like,for example, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, etc. Additionally, the peptide, polypeptideor variant thereof suspension may include lipid-protective agents whichprotect lipids against free-radical and lipid-peroxidative damages onstorage. Lipophilic free-radical quenchers, such as alpha-tocopherol andwater-soluble iron-specific chelators, such as ferrioxamine, aresuitable.

As used herein, a “surfactant” is a surface active agent which is agentsthat modify interfacial tension of water. Typically, surfactants haveone lipophilic and one hydrophilic group in the molecule. Broadly, thegroup includes soaps, detergents, emulsifiers, dispersing and wettingagents, and several groups of antiseptics. More specifically,surfactants include stearyltriethanolamine, sodium lauryl sulfate,sodium taurocholate, laurylaminopropionic acid, lecithin, benzalkoniumchloride, benzethonium chloride and glycerin monostearate; andhydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone,carboxymethylcellulose sodium, methylcellulose, hydroxymethylcellulose,hydroxyethylcellulose and hydroxypropylcellulose.

As used herein, “alkylglycoside” refers to any sugar joined by a linkageto any hydrophobic alkyl, as is known in the art. The hydrophobic alkylcan be chosen of any desired size, depending on the hydrophobicitydesired and the hydrophilicity of the saccharide moiety. In one aspect,the range of alkyl chains is from 9 to 24 carbon atoms; and further therange is from 10 to 14 carbon atoms.

As used herein, “Critical Micelle Concentration” or “CMC” is theconcentration of an amphiphilic component (alkylglycoside) in solutionat which the formation of micelles (spherical micelles, round rods,lamellar structures etc.) in the solution is initiated.

As used herein, “saccharide” is inclusive of monosaccharides,oligosaccharides or polysaccharides in straight chain or ring forms.Oligosaccharides are saccharides having two or more monosaccharideresidues.

As used herein, “sucrose esters” are sucrose esters of fatty acids.Sucrose esters can take many forms because of the eight hydroxyl groupsin sucrose available for reaction and the many fatty acid groups, fromacetate on up to larger, more bulky fats that can be reacted withsucrose. This flexibility means that many products and functionalitiescan be tailored, based on the fatty acid moiety used. Sucrose estershave food and non-food uses, especially as surfactants and emulsifiers,with growing applications in pharmaceuticals, cosmetics, detergents andfood additives. They are biodegradable, non-toxic and mild to the skin.

As used herein, a “suitable” alkylglycoside means one that fulfills thelimiting characteristics of the invention, i.e., that the alkylglycosidebe nontoxic and nonionic, and that it reduces the immunogenicity oraggregation of a compound when it is administered with the compound viathe ocular, nasal, nasolacrimal, sublingual, buccal, inhalation routesor by injection routes such as the subcutaneous, intramuscular, orintravenous routes. Suitable compounds can be determined using themethods set forth in the examples.

The terms peptide, polypeptide and protein may be used interchangeablyherein, or a peptide, polypeptide or variant thereof. As used herein,the term “polypeptide” is interpreted to mean a polymer composed ofamino acid residues, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof linked via peptidebonds, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.The term “protein” typically refers to large polypeptides. The term“peptide” typically refers to short polypeptides. “Polypeptide(s)”refers to any peptide or protein comprising two or more amino acidsjoined to each other by peptide bonds or modified peptide bonds.“Polypeptide(s)” refers to both short chains, commonly referred to aspeptides, oligopeptides and oligomers and to longer chains generallyreferred to as proteins. Polypeptides may contain amino acids other thanthe 20 gene encoded amino acids. “Polypeptide(s)” include those modifiedeither by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well-known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation, ADPribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-link formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1 12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626 646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 4862 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

As used herein, the term “agent” is interpreted to mean a chemicalcompound, a mixture of chemical compounds, a sample of undeterminedcomposition, a combinatorial small molecule array, a biologicalmacromolecule, a bacteriophage peptide display library, a bacteriophageantibody (e.g., scFv) display library, a polysome peptide displaylibrary, or an extract made from biological materials such as bacteria,plants, fungi, or animal cells or tissues. Suitable techniques involveselection of libraries of recombinant antibodies in phage or similarvectors. See, Huse et al. (1989) Science 246: 1275 1281; and Ward et al.(1989) Nature 341: 544 546. The protocol described by Huse is renderedmore efficient in combination with phage display technology. See, e.g.,Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047.

As used herein, the term “isolated” is interpreted to mean altered “bythe hand of man” from its natural state, i.e., if it occurs in nature,it has been changed or removed from its original environment, or both.For example, a polynucleotide or a polypeptide naturally present in aliving organism is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein.

As used herein, the term “variant” is interpreted to mean apolynucleotide or polypeptide that differs from a referencepolynucleotide or polypeptide respectively, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques, by direct synthesis,and by other recombinant methods known to skilled artisans.

The term “surfactant” comes from shortening the phrase “surface activeagent”. In pharmaceutical applications, surfactants are useful in liquidpharmaceutical formulations in which they serve a number of purposes,acting as emulsifiers, solubilizers, and wetting agents. Emulsifiersstabilize the aqueous solutions of lipophilic or partially lipophilicsubstances. Solubilizers increase the solubility of components ofpharmaceutical compositions increasing the concentration which can beachieved. A wetting agent is a chemical additive which reduces thesurface tension of a fluid, inducing it to spread readily on a surfaceto which it is applied, thus causing even “wetting” of the surface withthe fluids. Wetting agents provide a means for the liquid formulation toachieve intimate contact with the mucous membrane or other surface areaswith which the pharmaceutical formulation comes in contact.

While the effects of surfactants may be beneficial with respect to thephysical properties or performance of pharmaceutical preparations, theyare frequently irritating to the skin and other tissues and inparticular are irritating to mucosal membranes such as those found inthe nose, mouth, eye, vagina, rectum, buccal or sublingual areas, etc.Additionally, many and indeed most surfactants denature proteins thusdestroying their biological function. As a result, they are limited intheir applications. Since surfactants exert their effects above thecritical micelle concentration (CMC) surfactants with low CMC's aredesirable so that they may be utilized with effectiveness at lowconcentrations or in small amounts in pharmaceutical formulations.Typical alkylglycosides of the present invention have the CMC's lessthan 1 mM in pure water or in aqueous solutions. Some CMC values foralkylglycosides are listed below:

CMCs of some alkylglycosides in water:

Octyl maltoside 19.5 mM Decyl maltoside 1.8 mM Dodecyl β-D-maltoside0.17 mM Tridecyl maltoside 0.03 mM Tetradecyl maltoside 0.01 mM Sucrosedodecanoate 0.3 mM

The surfactants of the invention can also include a saccharide. As useherein, a “saccharide” is inclusive of monosaccharides, oligosaccharidesor polysaccharides in straight chain or ring forms, or a combinationthereof to form a saccharide chain. Oligosaccharides are saccharideshaving two or more monosaccharide residues. The saccharide can bechosen, for example, from any currently commercially availablesaccharide species or can be synthesized. Some examples of the manypossible saccharides to use include glucose, maltose, maltotriose,maltotetraose, sucrose and trehalose. Preferable saccharides includemaltose, sucrose and glucose.

The surfactants of the invention can likewise consist of a sucroseester. As used herein, “sucrose esters” are sucrose esters of fattyacids. Sucrose esters can take many forms because of the eight hydroxylgroups in sucrose available for reaction and the many fatty acid groups,from acetate on up to larger, more bulky fatty acids that can be reactedwith sucrose. This flexibility means that many products andfunctionalities can be tailored, based on the fatty acid moiety used.Sucrose esters have food and non-food uses, especially as surfactantsand emulsifiers, with growing applications in pharmaceuticals,cosmetics, detergents and food additives. They are biodegradable,non-toxic and mild to the skin.

While there are potentially many thousands of alkylglycosides which aresynthetically accessible, the alkylglycosides dodecyl, tridecyl andtetradecyl maltoside and sucrose dodecanoate, tridecanoate, andtetradecanoate are particularly useful since they possess desirably lowCMC's. Hence, the above examples are illustrative, but the list is notlimited to that described herein. Derivatives of the above compoundswhich fit the criteria of the claims should also be considered whenchoosing a glycoside. All of the compounds can be screened for efficacyfollowing the methods taught herein and in the examples.

In one embodiment of the invention, the present invention provides acomposition which reduces, prevents, or lessens peptide or proteinassociation or aggregation in the composition, for example, reducespeptide or protein self-association or self-aggregation, or reducesassociation or aggregation with other peptides or proteins whenadministered to the subject.

Self-Association at high protein concentration is problematic intherapeutic formulations. For example, self-association increases theviscosity of a concentrated monoclonal antibody in aqueous solution.Concentrated insulin preparations are inactivated by self aggregation.These self associating protein interactions, particularly at highprotein concentration, reduce, modulate or obliterate biologicalactivity of many therapeutics. Therapeutic proteins formulated at highconcentrations for delivery by injection or other means can bephysically unstable or become insoluble as a result of these proteininteractions.

A main challenge of protein formulation is to develop manufacturable andstable dosage forms. Physical stability properties, critical forprocessing and handling, are often poorly characterized and difficult topredict. A variety of physical instability phenomena are encounteredsuch as association, aggregation, crystallization and precipitation, asdetermined by protein interaction and solubility properties. Thisresults in several manufacturing, stability, analytical, and deliverychallenges.

Development of such formulations for protein drugs requiring high dosing(on the order of mg/kg) are required in many clinical situations. Forexample, using the SC route, approximately <1.5 mL is the allowableadministration volume. This may require >100 mg/mL proteinconcentrations to achieve adequate dosing. Similar considerations existin developing a high-concentration lyophilized formulation formonoclonal antibodies.

In general, higher protein concentrations permit smaller injectionvolume to be used which is very important for patient comfort,convenience, and compliance. Because injection is an uncomfortable modeof administration for many people, other means of administering peptidetherapeutics have been sought. Certain peptide and protein therapeuticsmaybe administered, by example, by intranasal administration. An exampleis calcitonin which is administered in a nasal spray. However there is alimit to the volume that can be practically dispensed into the nosewithout significant amount draining out.

Typical formulation parameters include selection of optimum solution pH,buffer, and stabilizing excipients. Additionally, lyophilized cakereconstitution is important for lyophilized or powdered formulations. Afurther and significant problem comprises changes in viscosity of theprotein formulation upon self association. Changes in viscosity cansignificantly alter delivery properties. This is perhaps most criticalin spray (aerosol) delivery for intranasal, pulmonary, or oral cavitysprays. Furthermore, increased viscosity can make injection delivery bysyringe or iv line more difficult or impossible.

Many peptide and protein molecules with useful therapeutic activity(hereafter called protein therapeutics) have been, and continued to be,discovered, therefore increasing the need for improved formulationtechnology. Examples include insulin, growth hormone, interferons,calcitonin, parathyroid hormone, and erythropoietin, among many others.Table I lists examples of peptide and protein therapeutics.

Table I. Examples of Peptide and Protein Therapeutics

TABLE I Examples of Peptide and Protein Therapeutics 1. Insulin likegrowth factor-I (IGF-I or Somatomedin-C) 2. Insulin 3. Calcitonin 4.Leptin 5. hGH 6. Human parathyroid hormone (PTH) 7. Melatonin 8. GLP-1or Glucagon-like peptide-1 9. GiP 10. OB-3 peptide 11. Pituitaryadenylate cyclase neuropeptide - activating polypeptide (PACAP) 12. GM-1ganglioside 13. Nerve growth factor (NGF), 14. D-tryp6)-LHRH 15.Nafarelin 16. FGF 17. VEGF. 18. VEGF antagonists 19. Leuprolide 20.Interferon-alpha 21. Interferon-beta 22. Interferon-gamma 23. Lowmolecular weight heparin 24. PYY 25. LHRH 26. LH 27. GDNF 28. G-CSF 29.Ghrelin antagonists 30. Ghrelin 31. KGF 32. IMITREX ™ 33. Integrelin 34.Nesiritide 35. SANDOSTATIN ™ 36. PTH (1-34) 37. desmopressin acetate 38.cetrorelix acetate 39. ganirelix acetate 40. bivalirudin 41. zafirlukast42. Exanitide 43. pramlintide acetate 44. Vasopressin 45. Desmopressin46. Glucagon 47. ACTH 48. GHRH and analogs 49. Oxytocin 50.corticotropin releasing hormone 51. TRHrh 52. Atrial natriuretic peptide53. Thyroxine releasing hormone 54. FSH 55. Prolactin 56. Tobramycin 57.Triptorelin 58. Goserelin 59. FUZEON ™ 60. HEMATIDE ™ 61. Buserelin 62.Octreotide 63. Gonadorelin 64. Felypressin 65. Deslorelin 66.Vasopressin, 8-L-Arg 67. Eptifibatide 68. GM-CSF 69. EPO 70.Interleukin-11 71. Endostatin 72. Angiostatin 73. N-acetyl oxyntomodulin30-37 74. Oxyntomodulin 75. Ularitide 76. Xerecept 77. Apo A-IV 78.rNAPc2 79. SECRETIN 80. Thymopentin 81. Neuromedin U 82. Neurotensin 83.Thrombospondin-1 inhibitors 84. FGF-18 85. FGF-20 86. FGF-21 87.Elcatonin Acetate 88. Antide Acetate 89. Dynorphin A (1-13) Acetate 90.Sincalide 91. Thymopentin Acetate 92. Thymosin alpha1 acetate(Thymalfasin) 93. Fertirelin Acetate 94. CRF Acetate 95. CRF (ovine) 96.Hisrelin 97. Thymalfasin 98. Ecallantide 99. Oxycortin 100. Urocortin101. ARIXTRA ™ 102. Spiegelmer nucleotide aptamers 103. CGRP (calcitoningene related protein) 104. Amylin 105. IL-21 106. melanotan 107.valpreotide 108. ACV-1 neuropathic pain peptide

Many attempts to stabilize and maintain the integrity and physiologicalactivity of proteins and peptides have been reported. Some attempts haveproduced stabilization against thermal denaturation and aggregation,particularly for insulin pump systems. Polymeric surfactants werestudied by Thurow and Geisen (1984) and Chawla et al., (1985) usedpolyol-surfactants. The stabilization of insulin by these compounds wasbelieved to be of a steric nature. Among other systems used aresaccharides (Arakawa and Timasheff, 1982), osmolytes, such as aminoacids (Arakawa and Timasheff, 1985), and water structure breakers, suchas urea (Sato et al., 1983). These compounds exert their action bymodulating the intramolecular hydrophobic interaction of the protein.

Hence, as used herein, the terms “association” or “aggregation” are usedinterchangeably. Protein association or aggregation is a common propertyof any polypeptide chain and the process can begin from at least apartially unfolded state. Peptide or protein aggregation can forminsoluble intracellular complexes, for example, amyloid plaques inneurodegenerative disorders. Peptide or protein aggregation can occurbetween one type or sub-types of class or family of peptides or proteinsor with another type or sub-type of a different class or family ofpeptides or proteins; the former is an example of peptide or protein“self-association” or “self-aggregation”.

Because many protein therapeutics undergo aggregation at highconcentration, it was desirable that a means be discovered to preventself association for the reasons mentioned above. Agents useful inpreventing self-aggregation of proteins at high concentrations orcontrolling viscosity must be essentially non-toxic and metabolized tonon-toxic products. Ideally, the agents should be physiologicallyoccurring substances or should metabolize to physiologically occurringmolecules and should not be subject to accumulation in the patients'tissues or organs.

Dodecyl maltoside has been demonstrated to prevent self-association ofinsulin and thus prevent inactivation of biological activity. However,various peptides, polypeptides, or proteins are encompassed in thepresent invention. Humanin peptides, a promising new class oftherapeutics, also aggregate thus limiting their biological activity,and investigators have had to resort to modifying the protein sequenceto reduce such aggregation.

Peptide T, and in particular its longer half-life analog D-Ala-PeptideT-amide (DAPTA), a very promising therapeutic for treatment of HIVinfection which has been shown to eliminate residual infectious virus inthe monocyte reservoir upon repeated administration, is subject to veryrapid aggregation and inactivation, thus limiting the usefulness (Ruffet al. 2001; Ruff et al. 2003; Polianova et al., 2003)

The Peptide T analog referred to as DAPTA is an octapeptide related tothe V2 region of HIV-1 gp120 and has been shown to be a non-toxic,CCR5HIV entry inhibitor that reduces plasma and persistently infected,treatment resistant macrophage reservoirs for at least six months. Thechemokine receptor CCR5 plays a crucial role in transmission of HIVisolates that predominate in the early and middle stages of infection aswell as those that populate the brain and cause neuro-AIDS. CCR5 istherefore an attractive therapeutic target for design of entryinhibitors.

Peptide T has a number of analogs. The most clinically useful is DAPTAwhich is D-ala¹-Peptide T-amide. However, other useful Peptide T analogsinclude: D-ala¹-Peptide T (lacks an amide at the C-terminus);D-ala¹Thr⁸-Peptide T amide; Vasoactive Intestinal peptide (VIP);Thr-Thr-Ser-Tyr-Thr (an active pentamer); and RANTES antagonists. RANTESis an octapeptide (Brain Research (1999) 838:27-36), and an acronym forRegulated on Activation, Normal T Expressed and Secreted. It is alsoknown as CCL5. RANTES is a cytokine that is a member of theinterleukin-8 superfamily of cytokines. RANTES is a protein. It is aselective attractant for memory T lymphocytes and monocytes. It binds toCCR5, a coreceptor of HIV. Blocking RANTES prevents HIV entry intocells.

Despite significant therapeutic successes major obstacles to a cureremain. The inability of current antiviral drugs to flush cellular viralreservoirs causes re-infection in the body. Toxicities, viralresistance, complicated regimens, and high cost greatly limit theeffectiveness of current therapies in the battle against global AIDS.

DAPTA has been clinically studied for almost 20 years and shown to becompletely non-toxic, effective in Phase I and placebo controlled phaseII NIH trials. This octapeptide is easy to manufacture and effective atvery low doses so that costs will be very low (less than $500 per year).It may be administered as a convenient nasal spray. The drug, which hasbeen tested with other antiviral regimens, is expected to havesynergistic treatment benefits without cross-tolerance and has beendemonstrated to be free of viral resistance for at least six months.

DAPTA has recently been proven to act as a receptor blocking entryinhibitor at CCR5 receptors (Polionova et al., 2005), a mechanism ofaction shown to be a highly desirable one for an HIV-1 therapy (Moore,2006). The HIV envelop (gp-120) derived Peptide T sequence was deducedlate in 1985 in a computer assisted database search for the part of thevirus which attaches to its receptor. Sophisticated knowledge of peptidereceptor pharmacology allowed the inventors, then at the NIH, to createa small long-lasting peptide therapeutic that blocked viral binding andinfection (Pert et al PNAS 1986).

In 1987, the report that DAPTA potently (10 pM) blocks envelop (gp120)binding and inhibits viral infectivity was met with vociferousobjections from the American HIV virological community which had failedto find a gp-120 receptor active peptide sequence after an extensivesearch. Objections, which were based on the failure to replicate DAPTA'santiviral effects in vitro, greatly diminished interest in clinicaltesting through the NIH/NIAD despite a report from Sweden (Wetterberg,et al., 1987) of dramatic improvements in four near terminal men withAIDS. The scientific controversy was resolved in 2001 with thedemonstration (Ruff et al., 2001) that DAPTA targets CCR5, not CxCR4chemokine co-receptors which prevailed in the Gallo lab-adapted strainin general use in 1987 and which is not representative of the HIVisolates that predominate in early HIV infection. The first report ofDAPTA's potent antiviral activity, 9 years before Peptide T chemokineco-receptors were known, had used Ruscetti's more physiological primaryisolate now realized to be a CCR5-using virus.

Between 1987 and 1990, Phase I clinical studies conducted by the NIMHwith some private funds showed a complete lack of toxicity, improvementsin peripheral neuropathies, and apparent positive benefits in NeuroAIDS,the focus of the NIMH. A phase II placebo-controlled NIMH trialconducted between 1990-1995 involved three sites, and 240 patients. This$11 M effort showed that DAPTA had significant clinical benefits versusplacebo for more cognitively impaired patients and a CD4 cell increasefell just short of statistical significance. A recent blind NIMHanalysis of virus levels in stored frozen plasma from this trialrecently revealed a significant (p<0.04) treatment effect.

In a trial of eleven persons (Polionova, et al., 2003) progressivelyless actual virus could be isolated from white blood cells and thetreatment-resistant persistently infected monocyte reservoir was greatlyreduced or flushed to undetectable levels in all patients. In a smallstudy, reversal of growth hormone secretion suppression has beenreported in children. In the last few years, analyses of the propertiesof formulated peptide and detailed structural studies (MacPhee,unpublished) have revealed the very strong tendency of DAPTA toaggregate upon storage resulting in the loss of both bioavailability andantiviral activity. It is now clear that this property of DAPTA hassometimes led to suboptimal clinical results (Simpson et al., 1996) andeven to falsely negative in vitro results. DAPTA has also been shown toresolve psoriatic lesions in an inflammatory skin disease (Raychaudhuriaet al., 1999).

Still, other reports describe the tendency of DAPTA to aggregate andform fibrils. For example, peptide T solutions have been reported tothicken and “gel”, potential loss of activity and/or the ability to betransported through the mucous membrane, e.g., the nasal epithelium, wasa consideration. Removing sodium chloride from the formulation andlowering the concentration to 5 mgs per mL appeared to solve theproblem. However, even at only 1 mg/mL, spectropolarimetric analysis atroom temperature revealed a shift from a large peak at the more dilute0.1 mg/ml of 205.4 nm to a large peak at 237.2 nm, indicating that thePeptide T was interacting with itself at higher concentrations inaggregation steps which would lead to gelation. Electron microscopyconfirmed that Peptide T formed fibrils, and to our best knowledgePeptide T forms fibrils more readily than any other small peptide yetdescribed (FIG. 1). In the present invention it has been discovered thatthis aggregation phenomenon results in loss of biological activity.Furthermore this tendency to aggregate or form fibrils not only variesfrom manufacturer to manufacturer but also varies unpredictably frombatch to batch as illustrated in the examples that follow.

Fibril formation is concentration and temperature dependent, withfibrils forming most rapidly at refrigerator temperatures andconcentrations at and above 1 mg/mL. For example, over many weeks ofstorage in the refrigerator, even 0.1 mg/mL peptide T solution graduallyand progressively lost substantially all ability to block HIV infection,as shown in the Examples below. Also, when a formulation of peptide T isstored for many months, e.g., in the refrigerator (about 4° C.), itshowed a 10-fold diminished ability to enter the plasma uponadministering via intra-nasal metered spray. The effects of fibrils wereconsidered so relevant and important that the Advanced ImmuniTy, Inc.(AITI), halted the clinical trials administering peptide T to treatpsoriasis.

Chronic neuroinflammation plays a prominent role in the progression ofAlzheimer's disease. Reactive microglia and astrocytes are observedwithin the hippocampus during the early stages of the disease.Epidemiological findings suggest that anti-inflammatory therapies mayslow the onset of Alzheimer's disease. Chemokine receptor 5 (CCR5)up-regulation may influence the recruitment and accumulation of glianear senile plaques; activated microglia express CCR5 and reactiveastrocytes express chemokines. Rosi, Pert and Ruff have previously shownthat neuroinflammation induced by chronic infusion of lipopolysaccharideinto the 4th ventricle reproduces many of the behavioral, neurochemical,electrophysiological and neuropathological changes associated withAlzheimer's disease (Pert et al. 2005).

In another embodiment, the present invention provides compositionshaving a peptide or protein drug and a surfactant having a CMC of lessthan about 1 mM, and preferably less than about 0.5 mM, that reduces orprevents aggregation while not denaturing the peptide or protein thusreducing or eliminating immunogenicity of the peptide or proteintherapeutic upon administration to a vertebrate, and which is notirritating but is nontoxic, either at the site of application orsystemically. Such a surfactant-peptide/protein drug composition isprovided herein.

In one embodiment, the present invention is based on the discovery thattherapeutic compositions comprising of least one self-associatingpeptide or protein drug and at least one surfactant, wherein thesurfactant is further comprised of at least one alkylglycoside, formstable, non-irritating formulations in which the aggregation of theself-aggregating protein or peptide is greatly reduced or eliminated,resulting in one or more benefits such as reduced or eliminatedimmunogenicity, reduced or eliminated loss of biological activityresulting from aggregation, a longer shelf life, or reduced cold chainrequirements as a result of reduction or elimination of inactivationupon spontaneous aggregation.

As used herein, “nontoxic” means that the alkylglycoside molecule has asufficiently low toxicity to be suitable for human administration andconsumption. Preferred alkylglycosides are nonirritating to the tissuesto which they are applied. Any alkylglycoside should be of minimal or notoxicity to the tissues, such as not to cause damage to the cell invivo. It is significant that the determination of toxicity be conductedin vivo, rather than in vitro. Much confusion and misinformationconcerning the relative toxicity of excipients exists. This is largelythe result of the currently unwarranted and uncritical reliance upon invitro testing methods. For example, recent studies directly comparing invitro and in vivo results have clearly demonstrated a lack ofcorrelation between in vitro and in vivo tests in predicting nasalirritation or toxicity. A well studied example is benzalkonium chloride(BAC). BAC has been used in nasal and ophthalmic products since 1935 atconcentrations up to 0.1%. However, over the past few years there havebeen conflicting reports of damage to human epithelia and exacerbationof rhinitis associated with products incorporating BAC.

In an extensive review and thorough analysis of the scientificpublications on this subject, Marple et al (Marple 2004) concluded thatthe current data indicate that any concerns raised were limited toresults from in vitro experiments. In direct contrast, analysis of thein vivo data suggested that even prolonged use of topical formulationscontaining BAC caused no significant damage to the nasal mucosa. Thedata analyzed were taken from 14 in vivo studies in which changes in thefunction and ultrastructure of nasal cilia were determined by varioustypes of microscopy including light microscopy, transmission electronmicroscopy, scanning electron microscopy, and inverted phase microscopy(Ainge 1994; Berg 1995; Braat 1995; Graf 1999; Holm 1998; Klossek 2001;McMahon 1997). Direct mucociliary clearance was evaluated viameasurement of indigo carmine saccharine transport time or saccharineclearance time and exacerbation of rhinitis was determined by changes innasal epithelia thickness. Likewise, in a well controlled double blindnasal biopsy study, 22 patients with perennial allergic rhinitisreceiving fluticasone propionate aqueous nasal spray containing eitherBAC, BAC plus placebo, or BAC alone for a six week period were studied(Braat 1995). There were no statistical differences betweenindigocarmine saccharine transport time and the number of ciliated cellspresent for each group, and scanning and transmission electronmicroscopy examination of the biopsied tissues showed no effects of BAC.

In another recent study examining nasal irritation caused bybenzalkonium chloride at 0.02%, saccharine transport time, anteriorrhinomanometry, determination of nasal secretions, orienting smell test,and anterior rhinoscopy showed no discernible negative effectswhatsoever (Lange 2004).

In a similar study by McMahon et al (McMahon 1997), conducted with 65normal volunteers over a two week period, no significant difference wasfound between subjects receiving nasal spray with or without BAC at0.02% twice a day on a double-blind basis. Symptoms scored includedacoustic rhinometry, saccharine clearance time, and ciliary beatfrequency. BAC caused a slight prolongation of mucosal ciliary clearanceafter application, but reportedly had no detectable effect on the nasalmucosal function after two weeks of continual regular use.

Another study which highlights the lack of correlation of in vitrotesting with in vivo experience in humans (Riechelmann 2004) and onewhich also offers a simple and plausible explanation of the lack ofcorrelation, the effect of the BAC on isolated nasal cilia taken from 15human donors was examined. In in vitro testing, BAC was seen to beciliotoxic. However, once again, in in vivo tests BAC did not altersaccharine transport time or indicators of proinflammatory effects,namely myeloperoxidase, and secretion of IL-6 and Substance P. Theauthors conclude that since no BAC-related proinflammatory effects areobserved that any ciliotoxic effect of BAC is probably neutralized bycomponents of secretions. This should not be too surprising since thisis essentially the function of the nasal secretions in the mucociliaryclearance process.

Thus it is clear that in vitro prediction of toxicity does not correlatewith actual in vivo experience in human subjects, and in vivo resultsare preferred in making such assessments.

Toxicity for any given alkylglycoside may vary with the concentration ofalkylglycoside used. It is also beneficial if the alkylglycoside chosenis metabolized or eliminated by the body and if this metabolism orelimination is done in a manner that will not be harmfully toxic.

In another embodiment of the invention, fluorinated organic solvents,polypeptide or variant thereof, or the peptide, polypeptide or variantthereof is admixed with a fluorinated organic solvent. The fluorinatedorganic solvent 2,2,2-trifluoroethanol (TFE) induces formation helicalcontent within peptide chains. For example TFE induces up to 48% helicalcontent within residues 1-20 of the peptide actin (Sonnichsen et al.,1992). Yet, another fluorinated organic solvent that induces structuralchanges within peptide chains is 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP).

Particular properties of TFE or HFIP make them ideal solvent forpeptides, polypeptides or variants thereof.

TFE and HFIP are commercially available in high purity. Thus, in anotheraspect of the invention, peptides, polypeptides and/or variants thereofcan be admixed with TFE or HFIP alone, or with any of the alkylglycosides described herein.

Many alkylglycosides can be synthesized by known procedures, i.e.,chemically, as described, e.g., in Rosevear et al., Biochemistry19:4108-4115 (1980) or Koeltzow and Urfer, J. Am. Oil Chem. Soc.,61:1651-1655 (1984), U.S. Pat. Nos. 3,219,656 and 3,839,318 orenzymatically, as described, e.g., in Li et al., J. Biol. Chem.,266:10723-10726 (1991) or Gopalan et al., J. Biol. Chem. 267:9629-9638(1992).

The linkage between the hydrophobic alkyl and the hydrophilic saccharidecan include, among other possibilities, a glycosidic, thioglycosidic(Horton), amide (Carbohydrates as Organic Raw Materials, F. W.Lichtenthaler ed., VCH Publishers, New York, 1991), ureide (AustrianPat. 386,414 (1988); Chem. Abstr. 110:137536p (1989); see Gruber, H. andGreber, G., “Reactive Sucrose Derivatives” in Carbohydrates as OrganicRaw Materials, pp. 95-116) or ester linkage (Sugar Esters: Preparationand Application, J. C. Colbert ed., (Noyes Data Corp., New Jersey),(1974)).

Examples from which useful alkylglycosides can be chosen for thetherapeutic composition include: alkylglycosides, such as octyl-,nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl-,hexadecyl-, heptadecyl-, and octadecyl-D-maltoside, -glucoside or-sucroside (i.e., sucrose ester) (synthesized according to Koeltzow andUrfer; Anatrace Inc., Maumee, Ohio; Calbiochem, San Diego, Calif.; FlukaChemie, Switzerland); alkyl thiomaltosides, such as heptyl, octyl,dodecyl-, tridecyl-, and tetradecyl-β-D-thiomaltoside (synthesizedaccording to Defaye, J. and Pederson, C., “Hydrogen Fluoride, Solventand Reagent for Carbohydrate Conversion Technology” in Carbohydrates asOrganic Raw Materials, 247-265 (F. W. Lichtenthaler, ed.) VCHPublishers, New York (1991); Ferenci, T., J. Bacteriol, 144:7-11(1980)); alkyl thioglucosides, such as heptyl- or octyl 1-thio β- orβ-D-glucopyranoside (Anatrace, Inc., Maumee, Ohio; see Saito, S, andTsuchiya, T. Chem. Pharm. Bull. 33:503-508 (1985)); alkyl thiosucroses(synthesized according to, for example, Binder, T. P. and Robyt, J. F.,Carbohydr. Res. 140:9-20 (1985)); alkyl maltotriosides (synthesizedaccording to Koeltzow and Urfer); long chain aliphatic carbonic acidamides of sucrose amino-alkyl ethers; (synthesized according to AustrianPatent 382,381 (1987); Chem. Abstr., 108:114719 (1988) and Gruber andGreber pp. 95-116); derivatives of palatinose and isomaltamine linked byamide linkage to an alkyl chain (synthesized according to Kunz, M.,“Sucrose-based Hydrophilic Building Blocks as Intermediates for theSynthesis of Surfactants and Polymers” in Carbohydrates as Organic RawMaterials, 127-153); derivatives of isomaltamine linked by urea to analkyl chain (synthesized according to Kunz); long chain aliphaticcarbonic acid ureides of sucrose amino-alkyl ethers (synthesizedaccording to Gruber and Greber, pp. 95-116); and long chain aliphaticcarbonic acid amides of sucrose amino-alkyl ethers (synthesizedaccording to Austrian Patent 382,381 (1987), Chem. Abstr., 108:114719(1988) and Gruber and Greber, pp. 95-116).

Some preferred glycosides include maltose, sucrose, and glucose linkedby glycosidic or ester linkage to an alkyl chain of 9, 10, 12, 13 or 14carbon atoms, e.g., nonyl-, decyl-, dodecyl- and tetradecyl sucroside,glucoside, and maltoside. These compositions are nontoxic, since theyare degraded to an alcohol or fatty acid and an oligosaccharide, andamphipathic.

The above examples are illustrative of the types of alkylglycosides tobe used in the methods claimed herein; the list is not exhaustive.Derivatives of the above compounds which fit the criteria of the claimsshould also be considered when choosing an alkylglycoside. All of thecompounds can be screened for efficacy following the methods taught inthe examples.

The compositions of the present invention comprising of at least onedrug and at least one surfactant, wherein the surfactant is furthercomprised of at least one alkylglycoside, can be administered in aformat selected from the group consisting of a drop, a spray, anaerosol, a lyophilizate, an injectable, and a sustained release format.The spray and the aerosol can be achieved through use of the appropriatedispenser. The lyophilizate may contain other compounds such asmannitol, gelatin, biocompatible gels or polymers. The sustained releaseformat can be an ocular insert, erodible microparticulates, swellingmucoadhesive particulates, pH sensitive microparticulates,nanoparticles/latex systems, ion-exchange resins and other polymericgels and implants (Ocusert, Alza Corp., California; Joshi, A., S. Pingand K. J. Himmelstein, Patent Application WO 91/19481).

The present invention mitigates and, in some cases, may eliminate theneed for organic solvents. Trehalose, lactose, and mannitol have beenused to prevent aggregation. Aggregation of an anti-IgE humanizedmonoclonal antibody was minimized by formulation with trehalose at orabove a molar ratio in the range of 300:1 to 500:1 (excipient:protein).However, the powders were excessively cohesive and unsuitable foraerosol administration or exhibited unwanted protein glycation duringstorage (Andya 1999). Each of the additives discovered have limitationsas additives to therapeutics including xenobiotic metabolism, irritationor toxicity, or high cost. The present invention provides excipientsthat are effective, non-irritating or toxic, do not require xenobioticmetabolism since they are comprised of the natural sugars, fatty acids,or long chain alcohols, and which may also be used to minimizeaggregation in aqueous solutions or upon aqueous reconstitution of driedpeptide or protein formulations in situ physiologic aqueousreconstitution by aqueous body fluids such as saliva.

In another embodiment, the invention provides methods of administeringto a subject in need thereof an effective amount of the therapeuticcompositions of the present invention. As used herein, “therapeuticallyeffective amount” is interchangeable with “effective amount” forpurposes herein, and is determined by such considerations as are knownin the art. The amount must be effective to achieve a desireddrug-mediated effect in the treated subjects suffering from the diseasethereof. A therapeutically effective amount also includes, but is notlimited to, appropriate measures selected by those skilled in the art,for example, improved survival rate, more rapid recovery, oramelioration, improvement or elimination of symptoms.

In one aspect of the present invention, a method of increasing theshelf-life of a drug composition by admixing a drug with a surfactantcomprising of at least one alkylglycoside and administering thecomposition to a vertebrate is described. As used herein, the phrase“shelf life” is broadly described as the length of time a product may bestored without becoming unsuitable for use or consumption. The “shelflife” of the composition described herein, can also indicate the lengthof time that corresponds to a tolerable loss in quality of thecomposition. The compositional shelf life as used herein isdistinguished from an expiration date; “shelf life” relates to thequality of the composition described herein, whereas “expiration date”relates more to manufacturing and testing requirements of thecomposition. For example, a composition that has passed its “expirationdate” may still be safe and effective, but optimal quality is no longerguaranteed by the manufacturer.

Shelf life is affected by light transmission, gas transmission, heattransmission, humidity transmission, or mechanical stresses. Nearly allchemical reactions will occur at common temperatures. These breakdownprocesses characteristically happen more quickly at higher temperatures.The usually quoted rule of thumb is that chemical reactions double theirrate for every 10 degree Celsius increase in temperature. The reason hasto do with activation energy barriers. Compositions described hereinallow the peptide or protein to retain at least twice the level ofbiological activity after storage at 25 degrees Celcius for at least onemonth. Other methods of increasing shelf life are known in the art andare encompassed in the present application in so much as they increasethe shelf life of the described compositions.

Another aspect of the invention provides light scattering as anon-destructive technique for characterizing the state ofmacromolecules. Light scattering can be routinely used to examine arange of macromolecules including their oligomeric (i.e., aggregated)states. Most importantly, light scattering permits measurement of thesolution properties of macromolecules. The intensity of the scatteredlight can measured as a function of angle or can be measured at fixedangles. For example a filter fluorometer in which the excitation lightpath is normally set at 90 degrees to the detection light path, and inwhich the filters are chosen to allow passage of the same lightwavelength, can be used as a convenient means to measure lightscattering. For the case of macromolecules, light scattering is oftencalled Rayleigh scattering and can yield the molar mass and rms radiusof the monomer or aggregate. Aggregates may vary widely in size up toformation of directly visible cloudiness (a light scattering phenomenon)or visible precipitates. Still, other methods including sedimentationequilibrium by ultracentrifugation may be used to observe aggregationdirectly.

In one embodiment, the present invention relates to a method forchemically modifying a molecule to increase or sustain the biologicalactivity of the composition or molecule, for example, receptor bindingor enzymatic activity. The molecule is preferably, although notnecessarily, a polypeptide. The method can include binding the moleculein the composition to a polymer such as polyethylene glycol.

The method(s) includes all aspects of the compositions described hereinincluding but not limited to compositions which reduced or eliminateimmunogenicity of peptide or protein drugs, are non-irritating, haveanti-bacterial activity, increased stability or bioavailability of adrug, decrease the bioavailability variance of that drug, avoid firstpass liver clearance and reduce or eliminate any adverse effects. Asused herein, the term “immunogenicity” is the ability of a particularsubstance or composition or agent to provoke an immune response. Theimmunogenicity of the peptides of the invention can be confirmed bymethods known in the art.

In another aspect of the present invention, a method of administering adrug composition comprising of at least one alkylglycoside mixed with atleast one drug and delivered to a vertebrate, wherein the alkyl has from9 to 24 carbon atoms, or further in the range of 10 to 14 carbon atoms,and the surfactant increases the stability and bioavailability of thedrug.

The methods of the present invention wherein the surfactant has a highNOAEL which is many times higher than the daily recommended intakeamount of that surfactant. For example, the NOAEL is from 10× to 1000×higher than the daily intake amount of the surfactant.

In another aspect of the present invention, a method of reducing oreliminating immunogenicity of a peptide or protein drug composition byadmixing the drug with a surfactant comprising of at least onealkylglycoside and/or sucrose ester, wherein the alkyl has from 10 to 14carbon atoms.

The methods of the present invention are delivered to a vertebratesubject in need of treatment including but not limited to, for example,a human. Moreover, depending on the condition being treated, thesetherapeutic compositions may be formulated and administered systemicallyor locally. Techniques for formulation and administration may be foundin the latest edition of “Remington's Pharmaceutical Sciences” (MackPublishing Co, Easton Pa.). Suitable routes may, for example, includeoral or transmucosal administration; such as intranasal; buccal;vaginal; rectal; as well as parenteral delivery, includingintramuscular, subcutaneous, intravenous, intraperitoneal, or intranasaladministration.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular subject in need of treatment maybe varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andlength of action of that compound, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular condition, and the hostundergoing therapy.

Other formulation components could include buffers and physiologicalsalts, non-toxic protease inhibitors such as aprotinin and soybeantrypsin inhibitor, alpha-1-antitrypsin, and protease-inactivatingmonoclonal antibodies, among others. Buffers could include organics suchas acetate, citrate, gluconate, fumarate, malate, polylysine,polyglutamate, chitosan, dextran sulfate, etc. or inorganics such asphosphate, and sulfate. Throughout this application, variouspublications are referenced. One skilled in the art will understand thatthe referenced disclosures of these publications are hereby incorporatedby reference into this application in order to more fully describe thestate of the art to which this invention pertains.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art. The following examples are intended to illustrate but notlimit the invention.

EXAMPLE 1 Insulin Compositions Having Reduced Immunogenicity

To six groups of three Sprague-Dawley rats (Charles River, Charlotte,N.C.) weighing between 300 and 350 grams each is administered either: 1)multiple intranasal (i.n.) doses of insulin in pH 6.0 in 5 mM sodiumacetate buffer, 0.9% saline, 0.18% dodecyl maltoside (Buffer A1) or0.125% sucrose monododecanoate (Buffer A2); 2) an intranasal controlcomprised of insulin in pH 6.0 in 5 mM sodium acetate buffer, 0.9%saline (i.e., containing no alkyl saccharide (Buffer B); 3) multiplesubcutaneous injections (s.c.) of insulin in Buffer A, and; or 4)multiple subcutaneous injections (s.c.) of insulin in Buffer B. Theintranasal and subcutaneous doses of insulin (0.5 U insulin per rat) areadministered once weekly and an equivalent amount (0.5 U) of insulin isadministered in a volume of 20 microliters intranasally or 100microliters by subcutaneous injection. A 3 mL aliquot of each of theabove solutions is lyophilized in a 21×70 mm amber 4 dram screw-top vialby first freezing the vials and contents and placing them in a LabconcoFreezon 4.5 lyophilizer in a Labconco 750 mL glass lyophilization vesselfor 36 hours.

Each rat is bled weekly for 12 weeks prior to the next administration ofinsulin. A 500 μl blood sample is drawn by orbital bleed into serumcapillary collection tubes. After blood collection, serum is preparedfrom each blood sample following coagulation by centrifugation of thecapillary tubes. All serum samples are stored at −70° C. prior toantibody determination.

Human insulin (recombinant, expressed in E. Coli, Sigma-Aldrich, St.Louis, Mo.) solutions prepared in pH 6 Na acetate buffer, 5 mM, 0.9%NaCl with (Buffers A) or without (Buffers B) 0.125% dodecyl maltoside(DDM) or sucrose dodecanoate (SDD). Insulin solutions are made on day 1of the study and stored thereafter at room temperature for the durationof the experiment.

For sample collection, rats are anesthetized with 2% Isoflurane in aPlexiglas anesthesia induction box to facilitate blood collection andinsulin administration.

Assay of anti-human insulin antibodies: Assay of anti-human insulinantibodies is conducted using Immunodiagnostic Systems Limited (IDS,Fountain Hills, Ariz.) anti-human insulin ELISA kit with themodification that the alkaline phosphatase labeled goat anti human IgGis replaced with alkaline phosphatase labeled goat anti-rat IgG(Sigma-Aldrich). Human insulin is immobilized onto microwells. Thepositive control, negative control, and diluted patient serum samplesare added to the appropriate microwells. Rat IgG antibodies specific tohuman insulin in the rat serum sample and controls bind to the insulinmolecules on the microwells. After washing off unreacted serummaterials, an enzyme (alkaline phosphatase) labeled goat antibodyspecific to rat IgG is added to the antigen-antibody complex. Afterthorough washing to remove the unbound enzyme, a substrate,para-nitrophenyl phosphate (PNPP), solution is added and the colordevelopment is scored visually. Two quality controls (positive andnegative) are provided to monitor and validate assay results. Noobserved color change in comparison to the negative control is scored as(−). Visible color development is scored on an increasing scale rangingfrom +/−, +, ++, +++. The intensity of the color is directlyproportional to the concentration of anti-insulin antibody.

No antibody is observed at the initiation of the study. After 2-3 weeks,antibody titers are seen to develop in the groups given thenon-alkylglycoside formulations. The titers increase over the subsequentweeks. See Tables II and III below. Based on relative ELISA titers, itis seen that formulations containing alkylglycosides result in lowerantibody responses.

Lyophilized formulations are reconstituted with 3 mL of water to givethe same concentration of drug as that prior to lyophilization. Uponadministration of the lyophilized and reconstituted formulations to asecond set of six groups of 3 rats per group and collection of bloodsamples as describe previously, the formulations containingalkylglycosides show essentially no immunogenicity whereas theformulations containing no alkylglycosides elicit a similar antibodyresponse to that seen in the non-lyophilized, non-alkylglycosidecontaining formulations. Thus lyophilization and reconstitution do notresult in increased immunogenicity in the presence of alkylglycosides,but do so in the absence of alkylglycosides

TABLE II Immunogenicity upon intranasal delivery of insulin in thepresence of dodecyl maltoside (DDM), an alkylglycoside No alkyl- Noalkyl- 0.18% DDM glycoside 0.18% DDM glycoside Buffer A1 Buffer B BufferA1 Buffer B (i.n.) (i.n.) (s.c.) (s.c.) Week Average Antibody Titers (n= 3 rats) 0 − − − − 1 − − − − 2 − − − + 3 − − − + 4 − + − ++ 5 − ++ + ++6 − ++ − ++ 7 + + − + 8 − ++ +/− +++ 9 − ++ − ++ 10 +/− +++ − +++ 11 −++ + ++ 12 − +++ − +++

TABLE III Intranasal delivery of insulin in the presence of sucrosemono-dodecanoate (SDD), an alkylglycoside No alkylglycoside Noalkylglycoside 0.125% SDD Buffer B (i.n.; 0.125% SDD Buffer B (s.c.;Buffer A2 same control Buffer A2 same control (i.n.) data as above)(s.c.) data as above) Week Antibody titers 0 − − − − 1 − − − − 2 − + − +3 − + − + 4 − + − ++ 5 − ++ +/− ++ 6 − ++ − ++ 7 +/− + − + 8 − ++ + +++9 − ++ − ++ 10 +/− +++ − +++ 11 − ++ + ++ 12 − +++ − +++

EXAMPLE 2 Insulin Alkyl Saccharide Compositions have Extended Shelf Life

The effectiveness of insulin formulations may be demonstrated in theOb-Ob mouse model of diabetes by performing a glucose tolerance test. Ina glucose tolerance test a bolus of glucose is administered to the Ob-Obdiabetic mouse by intraperitoneal injection. Because the animal isdiabetic, the glucose levels remain elevated for an extended period oftime. Upon intranasal administration of insulin (20 microliterscontaining 0.5 U, administered to a single nare) to the Ob-Ob mouse atthe time of the glucose bolus administration, blood glucose levels areseen to return to normal levels much sooner. As the insulin formulationages, insulin looses activity as a result of self aggregation. In thepresence of DDM and SDD, the insulin formulations are seen to retainactivity. See the Table below.

TABLE IV Insulin in the presence of alkylglycoside formulations haslonger activity Time (min) 0′ 15′ 30′ 45′ 60′ 90′ 120′ 180′ 240′ Bloodglucose levels, mg/dL No 190  430 495 370 320 270 218 172 170alkylglycoside T = 0 days No 190* 435 495 380 343 305 250 200 195alkylglycoside T = 28 days 0.125% DDM 190* 270 355 305 230 195 170 180180 T = 0 days 0.125% SDD 190* 275 340 310 225 190 170 175 170 T = 28days 0.18% DDM 190* 273 345 295 225 200 180 170 173 T = 28 days *Allsubsequent groups' initial glucose levels are normalized to 190 forintragroup comparison.

EXAMPLE 3 TFE Effectively Reduces Fibril Formation and Aggregation

In one embodiment of the invention, there is provided methods to preparepeptide T or analogs thereof, e.g., D-Ala-Peptide T-amide (DAPTA)solutions. In one aspect of the invention, the peptide T or analogsthereof, are of high potency, or bioactivity, and free from fibrils. Thefibril formation is, in part, dependent upon salt, temperature,manufacturing, and peptide concentration. However, other physiochemicalelements which contribute to fibril formation are contemplated. Thefollowing describes a method for reducing or inhibiting fibril formationin peptide T and/or analogs thereof. The methods described hereinprovide for peptide T and/or analog formulations thereof that are10-fold greater in potency and bioactivity than peptide T and/or analogformulations in the absence of such conditions or medium. For example,the peptide T or analog formulations thereof, have improved or enhancedor increased blood concentration of the peptide, e.g., increased bloodconcentrations of DAPTA.

Circular dichroism (CD) studies show that there is thresholdconcentration near or about or below 0.1 mM, whereby the rate of fibrilformation is greatly reduced. The peptide T or analog formulationsdescribed herein involve but are not limited to adjusting the DAPTAconcentration to be near or below 0.1 mM,

Additionally, it has been determined that deleting the NaCl, commonlyused in government and industry trial formulations, greatly inhibitsgellation of peptide T formulations. The mixing of the aqueous/alcoholsolution with the solid peptide T occurs immediately (i.e. before thefirst application). The mixing can occur in a bottle or device designedto allow mixing and holding enough solution for a short period of time,e.g., less than 1 day, less than one week, less than two weeks, lessthan three weeks and the like. Once reconstituted, the peptide Tformulation, or drug, should be at room, or ambient temperature.

The fibril formation process is thought to initiate from a slowlyforming nucleation seed, which is poorly defined. However, onceinitiated, extension and then stacking can proceed much more rapidly.Hence, one aspect of the methods described herein is to remove anynucleation seeds, thus preventing fibril formation. The nucleation seedscan be various contaminants or nascent aggregates from the manufacturingprocess, for example, lyophilizing the peptide. Different manufacturingprocesses or even unpredictable and uncontrolled batch to batchvariability in the same manufacturing process may yield more or lessnucleation seeds as illustrated in the examples which follow. Variousmethods to remove the contaminants or aggregates include, but are notlimited to, micro-filtration, ultrafiltration, affinity chromatography,selective absorption chromatography, ion exchange chromatography,lyophilization, dialysis, and precipitation or salting-out.

Still, the methods described herein encompass those solvents whichdisrupt peptide fibril or aggregate structure, for example, by inducingformation of alpha-helixes, and thereby removing or preventing fibrilformation. Trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP) have been shown to have this property for amyloids and otherpeptides. The invention also encompasses various variants, mutations(e.g., deletions) which will stabilize a peptide and prevent fibrilformation. For example, the so-called amyloid-β (e.g., 1-42 residues)peptide associated with Alzheimer's disease is highly fibrillogenic,while peptides lacking residues 14-23 are not (Tjernberg et al., 1999,J. Biol. Chem. 274:12619-12625). Similarly, peptide T and/or analogsthereof, including DAPTA, may therefore contain deletions and/ormutations as compared to the wild-type sequence which stabilizes fibrilsand or inhibits, reduces or prevents fibril formation.

Moreover, the methods of peptide T or analog formulation describedherein may undergo various filtration steps and additional admixingsteps with solvents prior to a final finishing step or a finallyophilization from solvents. The method described herein can alsoencompass additional steps such as modifying pH, addition of salts, etcwhich block or remove nucleation seeds. The method described herein canutilize an antibody or a peptide or agent which stabilizes the unstableor discordant helix, or specific region of a discordant helix by bindingto that region and allow for stability. Identified substances are thentested for their ability to inhibit fibril formation, e.g., stabilizeα-helical conformation. Another approach to identifying compounds thatinhibit fibril formation and/or stabilize the α-helical conformation isto screen chemical libraries for molecules that inhibit fibril formationand stabilize an α-helical conformation using methods such as thosedescribed herein. Thus, methods described herein encompass an assay fordetection of fibril formation of a drug, or peptide T or analog thereof,e.g., DAPTA, in the presence and absence of a test compound, e.g., acompound identified from a chemical library above, to prescreen testcompounds for those that are to be used in subsequent assays of α-helixstabilization. Similarly, the ability of a candidate compound to inhibitfibril formation can be used to confirm the predicted efficacy of acandidate compound in preventing fibril formation.

Concentrations of 5 mgs per mL, 0.5 mgs per mL, and 0.05 mgs per mL andbelow with or without the addition of GRAS (i.e., so-called in FDAregulations as Generally Recognized As Safe) reagents such as but notlimited to EDTA, buffers, preservatives, chelators, and the like, aswell as alkyl glycosides and/or alkyl saccharides described may be usedto further suppress and prevent fibril formation. Simple sugars, byvirtue of their alcoholic groups (—OH) may disrupt bonding leading tostacking as the DAPTA peptide is rich in threonines which contain (—OH)groups. Modifications in the peptide primary sequence, or side groups toreduce intermolecular bonding, would be useful, and are contemplated.These improvements will enhance potency, requiring less drug to beadministered, and extend the useful storage period of the drug.

Fibril formation can be monitored by examining the spectropolarimetricshift, elecronmicroscopy studies, and/or other methods, e.g.,dye-binding techniques, as described herein. Additionally, biologicaltesting, for specific activity, as an antiviral (Ruff, M R, et al.,2001), chemo-attractant (Redwine, 1999), agonist or antagonist ofMAPkinases (Ruff, pert, Meucci, unpublished data), or transcriptionfactors, for example, can also be used.

Biophysical studies revealed that DAPTA has a tendency to form fibrillaraggregates in aqueous solutions, similar or identical to those used inthe formulation of DAPTA in prior clinical trials. These fibrillaraggregates are biologically inactive, and would be expected to havedistinctly different pharmacokinetic and pharmacodynamic properties fromthe monomer. The detailed study of the DAPTA aggregates and DAPTA fibrilformation in aqueous solutions is described herein.

Peptide Storage. Peptides were stored at −20 deg. C. as dry powders,from the stated dates of synthesis.

Preparation of DAPTA Solutions. DAPTA was Dissolved in Water, andsolutions maintained at various temperatures and times. To preparefibrils, DAPTA was prepared at 10 mgs/mL in water and stored overnightat 4° C.

Determining fibril formation. Fibril formation of peptide T or analogthereof can be determined using electron microscopy. A 2 μl aliquot ofthe DAPTA solution in water was applied to a formvar/carbon coatednickel EM grid. The grids were rinsed ×3 with 10 μl distilled water andstained with 10 μl of 2% uranyl acetate. The samples were examined on anFEI TEM Tecnai microscope with a LaB6 filament (120 kv) and imaged witha Megaview II CCD camera.

Fibril formation of peptide T or analog thereof can also be determinedusing dye binding. Congo red was dissolved in PBS (5 mM potassiumphosphate, 150 mM NaCl, pH 7.4) to a concentration of 7 ug/mL. Thesolution was chilled to 4° C., and DAPTA added as a 10 mg/mL stocksolution in water, to yield final peptide concentration in the dyesolution of 0.48 mg/mL. Peptide solution immediately after dissolutionof powder was compared with an aged stock solution containing aggregatedpeptides. Spectra were collected between 400-700 nm, at 4° C.

Fibril formation of peptide T or analog thereof can also be determinedusing Circular Dichroism (CD) spectroscopy. Ten mg/mL solutions in waterof either freshly prepared peptide or containing fibrillar aggregateswas added to distilled water at 4° C. to a concentration of 50 ug/mL. CDspectra were collected on a Jasco model J-810 spectrometer using a 0.1cm path length quartz cuvette, between 190-250 nm, with a 1 mininterval, and a response time of 2 sec.

Still another method of determining fibril formation of peptide T oranalog thereof is performed using Fourier Transform Infrared (FTIR)Spectroscopy. DAPTA was dissolved in deuterated water, to aconcentration of 10 mg/ml and incubated under temp and time conditionsthat promote fibril formation. 25 ul samples were then placed in apre-cooled transmission cell with NaCl windows separated by a 6 umspacer. FTIR spectra were collected on a BioRad FTS-175C Fouriertransform spectrometer in transmission mode using a DTGS detector. 2506interferograms were recorded with a 2 cm-1 resolution. Water vapor wassubtracted and the spectra baselines corrected.

The results were as follows. Peptide T or an analog thereof, e.g.,DAPTA, aggregates in solution, in some cases into well-ordered bundles.Fibril formation has been followed by the various techniques describedherein (e.g., EM, FTIR, CD and dye binding). Preliminary X-raydiffraction studies suggest that ordered fibrillar aggregates arecomposed of peptides, packed in narrow parallel arrays of B sheets, andstacked perpendicular to the long axis of the fibril (FIG. 1).

In solution, DAPTA can be shown to form ordered aggregates by EM, FTIR,and CD and dye binding. Aggregation is promoted by concentration,increased ionic strength, and reduced temperature. Although the kineticsof aggregation appear to vary, from preparation to preparation,aggregation appears to be a property associated with all batches ofDAPTA examined as measured by EM.

Fibrillization can be observed in solutions prepared in 0.9% saline (10mg/mL) when stored at 4° C. in less than 1 hour. At room or ambienttemperature, fibril formation can be observed within 48 hours, althoughthere is variation from preparation to preparation. In distilled water,DAPTA solutions at 10 mg/ml readily form aggregates at 4 deg. C., within2 hrs, and at room temperature, within 1-7 days.

Aggregation is discovered to be associated with a loss of biologicalactivity in vitro. Typically, the recommended protocol is to have DAPTAstored at 0.1 mM solutions in water at 4° C. However, DAPTA under theseconditions was found to form fibrils as observed and confirmed by EM anddisclosed herein. DAPTA stored under these conditions was observed tohave reduced activity event though the chemical integrity of the peptideappears unchanged as measured by HPLC; and by about 6 weeks, the DAPTAformulation exhibits substantial loss of activity as measured by HIVuptake inhibition in vitro as disclosed in the Examples which follow.Thus, currently recommended protocols, which maintain DAPTA at 0.1 mMconcentration solutions or lower, at 4° C., create peptide aggregatesand form inactive preparations.

In one embodiment, aqueous solutions of aggregated peptides can bepartially dissociated by warming the peptide aggregates, for example,DAPTA solutions at 5 mg/mL in 0.45% NaCl reversibly dissociate whensolutions are warmed to 37° C., for about 17-24 hours, e.g., 17-18 hourswith shaking. The experiment was done in parallel except shaking wasperformed at room or ambient temperature. Treatment with TFE over timeand heat drives substantially all DAPTA into an alpha-helicalconformation and out of β-sheet forms, which DAPTA favors, and hencedissociates DAPTA into monomers. Thus, substantially all aggregationseeds having at least 2, or 2 or more molecules of DAPTA to formβ-sheets. Following reduction or inhibition of β-sheet formation, theDAPTA solution can then be lyophilized without aggregation. Thelyophilized peptide can then be reconstituted in water and the like, andis capable of being stored in water for an extended period of time.

Aggregation is reduced in the presence of trifluoroethanol (TFE). TFEwas selected because of its property of reducing certain types ofprotein-protein interactions. DAPTA was dissolved in either distilledwater, or solutions containing between 60% and 100% TFE. Aggregation wasevaluated by assaying inhibition of HIV infectivity, in vitro. DAPTAstored in solution with TFE at concentrations between 60% and 100%retained more activity as compared to DAPTA stored in the absence of TFEor in water under equivalent conditions. Also, TFE is capable ofdisassociating preformed aggregates of DAPTA. Aggregates of DAPTA,formed in distilled water were disrupted by addition of TFE to 80%, asmeasured by EM.

EXAMPLE 4 DAPTA Compositions with TFE and/or Alkyl Glycosides haveIncreased Bioactivity and Extended Shelf Life

In “in vitro” studies, DAPTA it has been reported to prevent HIV frominfecting CD4 cells by blocking receptor sites on the CD4 molecule(Bridge et al. 1989; Pert and Ruff 1986; Pert et al. 1988; Ruff et al.1987; Ruff et al. 1991). DAPTA is an octa-peptide which mimics andcompetes with both a section of VIP and a section of gp120, the HIVsurface molecule which binds to the CD4 receptor. Brenneman et al.(1998) reported that DAPTA and VIP can prevent gp120-induced neuronalcell death “in vitro”. Simpson et al. (1996) reported that in a phase IIdouble-blind efficacy trial of DAPTA, there were no statisticallysignificant differences between DAPTA (6 mg/day for 12 weeks) andplacebo in the treatment of painful peripheral neuropathy.

The drug also seemed to have no effects on neuropsychological functions.The study enrolled 81 participants with AIDS. Heseltine et al., (1998)treated 215 people with mild to severe cognitive impairment with eitherDAPTA (2 mg three times daily intranasally) or placebo for six months,followed by open-label DAPTA for an additional six months. Analysis ofall people who completed at least four months of treatment showed therewas no difference in neuropsychological performance between the twoarms. After the analyses were adjusted to take account of an imbalancein baseline CD4 count between the groups, people who received DAPTAshowed greater improvement (p=0.07). In particular, DAPTA was beneficialfor people with CD4 counts greater than 200 or with more evidentcognitive impairment at baseline. Those with a baseline deficit scoreabove 0.5 showed overall cognitive improvements while the placebo groupexperienced an overall deterioration in cognitive performance. Kostenconducted a placebo-controlled, double-blind, cross-over study of 15 mgor 1.5 mg of DAPTA daily in nine injecting drug users with early AIDSdementia. Neuro-psychological performance improved in 4/5 patients whoreceived high dose DAPTA compared to only ¼ in the low dose group(Kosten et al., 1997). Participants were also receiving methadone andAZT monotherapy. Bridge et al. (1989) reported a phase I safety anddosing study of DAPTA in 14 people with AIDS. Drug was dosed from 0.1 to3.2 mg/kg/day intravenously for twelve weeks. The first six patients tocomplete treatment continued on intranasal drug (25 mg/day for eightweeks). Cognitive and neuromotor function improved in patients withmoderate neuro-psychological impairment compared with controls.MacFadden and Doob (1991) reported that of nine individuals withHIV-related peripheral neuropathy treated with DAPTA (subcutaneously atan initial dose of 10 mg daily, with two patients tapered to 2.5 mg inorder to determine the minimal effective dose), all experienced eithercomplete or subjectively significant resolution of lower limb pain, witheffects being noticed as early as two days after initiation oftreatment. The pain-free interval persisted for the duration of thetreatment (for 3 to 70 weeks) but pain recurred gradually within oneweek of stopping the drug, resolving upon reinstitution of treatment. In2 participants, decreasing the dose to 2.5 mg/day resulted in recurrenceof pain, which resolved when the dose was increased to 5 mg. No adversedrug effects were noted.

Cultured monocytes were infected with the SF-163 strain of HIV. Thelevel of P24 antigen was measured in the cell supernatant and is anindication of the presence of infectious virus. In the controldesignated as virus only control, the concentration of P24 antigen isapproximately 154.5 picograms per mL. DAPTA has been seen to aggregaterelatively quickly resulting in a significant to nearly complete loss ofactivity. Thus samples 13, 14 and 15, which were aged for seven daysbefore use, exhibit concentrations of P24 antigen similar to that seenfor the virus-only control and thus have essentially no activity. Whensolutions of DAPTA are prepared in the presence of 80% trifluoroethanol,the solutions remain active for an extended period of time as seen bythe reduced levels of P24 antigen. Unfortunately, trifluoroethanol isnot a desirable solvent for use in a therapeutic formulation. Whendodecyl maltoside or sucrose mono-dodecanoate is added to solutions ofDAPTA, the activity is seen to remain for an extended period of time,once again as seen by the reduced levels of P24 antigen. Concentrationsof dodecyl maltoside or sucrose mono-dodecanoate used in this experimentwere approximately 0125% to 0.2% per mL. The alkyl saccharidessignificantly stabilize DAPTA by preventing aggregation and thusincrease the shelf life of this very promising anti-HIV therapeutic. Seethe Table below.

In another embodiment of the invention, DAPTA formulations were admixedin 80% TFE and shaken at 37° C. for about 17-18 hours. The formulationswere lyophilized using speedvac, and stored as a lyophilized powderuntil dissolved in H₂0 with or without alkylglycosides. Theseexperiments demonstrate that in the presence of the surfactantsdescribed herein, e.g., alkyl glycosides such as dodecyl maltoside (DDM)or sucrose mono-dodecanoate (SDD), there is a significant improvementwith regards to reduction of peptide aggregation as compared to parallelstudies in the absence of the surfactants.

In one aspect of the invention, TFE can be introduced to DAPTA as a nearlast step, and the solvent evaporated.

Thus, the invention described herein demonstrates that syntheticpreparations of peptide T, e.g., DAPTA, independent of source and dateof synthesis, form aggregates as confirmed by spectroscopic methods,e.g., X-ray diffraction, and direct visualization by EM. These peptideaggregates are promoted by increasing peptide concentration, decreasingtemperature, increased ionic strength and is time dependent (hours). Thein vitro studies show that peptide aggregation reduces the biologicalactivity of the peptide, polypeptide or variant thereof, e.g., DAPTA.Further that use of co-solvents such as TFE or HFIP reduces theformation of aggregates and disrupts preformed aggregates. Lastly, theordered structure of the peptide aggregates suggests that specificinteractions are responsible. Therefore, although TFE is not generallyincluded in pharmaceutical preparations and or therapeutic compositions,its properties lend themselves to the invention described herein. Still,other excipients or agents or co-agents can be included in the peptidetherapeutic formulation to inhibit fibril formation or prevent or reducethe aggregation formation.

TABLE V DAPTA in the presence of alkylglycosides extends stability anddrug shelf-life Samples ID Concentration of DAPTA p24Ag pg/mL Mean StdDev No TFE - aged 7 days before use 1 2.5 mg/mL in DDM 131.104 93.545112.325 18.78 2 0.5 mg/mL in DDM 43.208 36.435 39.822 3.39 3 0.05 mg/mLin DDM 69.993 61.372 65.683 4.3 No TFE - aged 7 days before use 4 2.5mg/mL in SDD 41.361 34.588 37.975 3.4 5 0.5 mg/mL in SDD 56.601 54.59955.6 1.00 6 0.05 mg/mL in SDD 18.887 22.735 20.811 1.9 From 80% TFE -aged 7 days before use 7 2.5 mg/mL in H₂O 70.301 67.838 69.07 1.2 8 0.5mg/mL in H₂O 70.608 75.073 72.84 2.2 9 0.05 mg/mL in H₂O 63.22 61.21962.2 1 Samples in H₂O - made fresh at time of use 10 2.5 mg/mL madefresh 12.729 8.727 10.728 2 11 0.5 mg/mL made fresh 154.656 148.807151.73 2.9 12 0.05 mg/mL made fresh 100.625 329.063 Outlier Samples inH₂O - aged 7 days before use 13 2.5 mg/mL in H₂O 160.66 100.01 130.33530.3 14 0.5 mg/mL in H₂O 96.931 65.837 81.384 15.5 15 0.05 mg/mL in H₂O168.51 177.284 172.897 4.4 Virus only 1:10 154.502 134.029 144.27 10.2Non treated cells only −30.218 −32.681 0

EXAMPLE 5 Quantitative Measurement of Protein Stabilization byAlkylsaccharides Using Light Scattering Measurements

This study was performed to determine and document the effects ofalkylsaccharide surfactants described herein on the aggregation ofvarious proteins in solution at 37° C. at varying pHs. Recombinant humaninsulin (Humulin-R, manufactured by Eli Lilly) and human growth hormoneor hGH (Humatrope, manufactured by Eli Lilly) solutions containingalkylsaccharides were prepared, along with identical control proteinsolutions without alkylsaccharides. Solutions were incubated at 37° C.on a rotary platform shaker (LabLine thermoregulated shaker) at 150 rpmfor up to three weeks. Protein aggregation was determined bymeasurements of light scatter using a Shimadzu RF-500 recordingspectrofluorophotometer with both the excitation and emissionwavelengths set at 500 nm. Measurements were taken on Day 0 and atvarious time intervals during the three week period.

Insulin preparations. 25 ml solutions of Humulin-R (insulin) at 0.5mg/ml and lysozyme at 1.0 mg/ml were prepared in citrate buffer at pH5.5, 6.5 and 7.4, without and with dodecyl maltoside and sucrosedodecanoate at 0.250%, 0.125% and 0.062% final surfactantconcentrations, by dilution of Humulin-R U-100 (Lilly HI-210, 100units/ml) recombinant human insulin stock solution at 4.0 mg/ml protein.The final buffer composition was: 5 mM Citric Acid+0.1% EDTA, titratedwith NaOH to pH 5.5, 6.5 and 7.4. Each solution was stored in a 50 mlglass vial and capped with parafilm. Day 0 light scatter measurementswere performed on the insulin samples at pH 6.5 and 7.4, and then thesamples were re-sealed with parafilm and incubated at 37° C. with 150rpm shaking (FIGS. 2 and 3).

Human Growth Hormone (hGH) preparations. Humatrope human growth hormone(hGH) 5 mg lyophilized. The vial of Humatrope was dissolved in 9.0 ml ofbuffer, then split into two 10 ml glass vials and stored overnight at 4°C. Solubility was good. Buffer was added to the control vial to a finalvolume of 5 ml. Buffer and dodecyl maltoside from a stock solution wereadded to the second vial to a final concentration of 0.125% and a finalvolume of 5 ml. The final buffer composition in each vial was: 5 mMCitric Acid+0.1% EDTA, titrated with NaOH to pH 6.5. The correspondingcontrol solution contained no dodecyl maltoside. Day 0 light scattermeasurements were performed on the two hGH samples, and then the sampleswere re-sealed with parafilm and incubated at 37° C. with 150 rpmshaking (FIG. 4).

Light Scatter Measurements. Light scatter was measured for each proteinsample at selected time points during the three week study with aspectrofluorophotometer (Shimadzu model RF-1501). Both excitation andemission wavelengths were set to 500 nm, and samples were read indisposable cuvettes with a 1 cm path length. For each reading, theinstrument was zeroed with 1 ml of the appropriate buffer, then analiquot of protein sample was added, mixed by inverting multiple times,and the cuvette was checked for air bubbles before three stable readingswere recorded. The spectrofluorophotometer was set for high sensitivityand the maximum possible reading was 1000 units. Insulin samples at Day0 were measured with 50 ul aliquots, then with 10 μl aliquots forreadings at subsequent time points. Light scatter measurements for thetwo hGH samples used 5 μl and 10 μl aliquots at Day 0 and at each timepoint. After light scatter readings were taken, each protein sample wasre-sealed with parafilm and returned to 37° C. with 150 rpm shaking. Theresults are shown in the Table below and FIGS. 2, 3 and 4. Results forinsulin at pH 5.5 were essentially the same as pH 6.5 over the 20 dayperiod. In each case, “A” designates dodecyl maltoside; “B” designatessucrose dodecanoate.

TABLE VI Insulin light scatter measurements (Average of 3 readings) Day0 Day 1 Day 2 Day 6 Day 9 Day 13 Day 16 Day 20 Insulin pH 6.5 Control84.8 589.1 1003 1002 1002 1002 1003 1005 0.062% A 89.2 28.6 3.6 9.7 9.37.0 11.7 18.2 0.125% A 147.3 9.0 10.5 12.5 8.7 9.9 5.3 3.7 0.250% A 84.13.7 16.0 4.1 7.5 15.0 7.0 6.5 0.062% B 71.3 30.3 11.2 5.3 9.2 7.6 11.414.8 0.125% B 39.1 18.5 10.7 7.8 5.8 18.1 13.2 9.3 0.250% B 15.8 8.7 4.515.0 14.9 26.2 19.8 16.3 Insulin pH 7.4 Control 69.6 993.9 1003 10041003 1003 1004 1000 0.062% A 106.1 18.7 5.5 10.3 10.0 6.8 17.0 — 0.125%A 104.0 10.2 10.7 11.6 8.0 12.0 — — 0.250% A 100.2 20.0 6.5 28.6 — — — —0.062% B 57.9 16.5 2.8 7.1 11.6 7.3 8.4 4.5 0.125% B 77.7 11.5 7.0 14.711.2 11.6 7.6 10.8 0.250% B 32.4 11.8 7.8 26.9 22.3 10.7 14.5 29.3 A =dodecyl maltoside; B = sucrose dodecanoate

TABLE VII hGH light scatter measurements (Average of 3 readings at 5 μLand 10 μL sample sizes) hGH pH 6.5 Day 0 Day 1 Day 2 Day 3 Day 7 Day 10Day 13 Day 16 Day 20 Control 5 μl 76.1 105.8 114.3 105.2 103.6 97.2 99.1108.7 110.7 0.125% A 5 μl 48.0 21.4 18.5 10.2 27.5 18.2 13.9 17.7 7.3Control 10 μl 353.2 241.4 175.6 197.1 241.5 254.6 314.1 304.7 216.40.125% A 10 μl 109.7 26.4 30.9 31.0 26.9 17.0 18.1 16.0 17.5 A = dodecylmaltoside

EXAMPLE 6 Tendency of Various Stored Powdered Samples of D-ALA Peptide TAmide (DAPTA) to Form Fibrils

Peptides were stored at −20° C. as dry powders, from the stated dates ofsynthesis, then dissolved in water at a concentration of 10 mgs/ml whichhas been used in many clinical trials, and solutions maintained atvarious temperatures and times. Samples were examined by ElectronMicroscopy using a 2 μl aliquot of the Dapta solution in 0.9% salineapplied to a formvar/carbon coated nickel EM grid. The grids were rinsed×3 with 10 μl distilled water and stained with 10 μl of 2% uranylacetate. The samples were examined on a FEI TEM Tecnai microscope with aLaB6 filament (120 kv) and imaged with a Megaview II CCD camera. By thismethod, fibrils were most easily and reliably visualized. Of 100 fieldsexamined, +++++ means that fibrils were most readily detected while +means fibrils were rarely detected.

Results:

Phoenix Pharmaceuticals

-   -   April 2003    -   Code: 057-03    -   Lot #: 20569 +++++        Peptech (Europe)—Denmark    -   February 1995    -   a) lot #171101, product #3022 +    -   b) Lot #17543 ++        Calbiotech    -   March 1995    -   a) Lot #101601 ++    -   b) Lot #101801 +        Peptide Technologies Corp.    -   3-20 +++        Penninsula Labs    -   a) GMP #539, Lot #036299 +++    -   b) Code #9301, Lot #036299 +++    -   c) Mar. 9, 1995, Lot #022376 ++++    -   d) Code #7444, Lot #801688+++

EXAMPLE 7 DAPTA Time Dependent Loss of Anti-Viral Activity Upon Storagein Solution

Aqueous solutions of DAPTA comparable to clinical formulations (0.1 mMin water) were prepared and their biological potency tested afterstorage at ambient temperatures for various times. DAPTA was synthesized(Peninsula, Belmont, Calif., 95% pure). Peptide was dissolved at 5 mg/mlin water, stored at ambient temperature, ca 23-27° C., and samplestested for biological activity in an HIV infection assay.

Inhibition of HIV infection is studied by utilizing an infection assayin which GHOST CD4 CCR5 cells are infected with HIV BaL, an R5 tropicisolate (Ruff, M R et al., 2001). Infection is detected via viralinduction of the hGFP gene (green fluorescent protein) 48 hrspost-infection. Trays are measured in a plate reader to determinefluorescence intensity. All infections are performed by adding freshculture medium containing approximately 1,000 infectious units of HIV-1per well (96-well plates).

Samples were then aged by storage at ambient temperature (ca 25° C.) for14 days prior to antiviral testing. The results are shown FIG. 5. InFIG. 5, only the short term 6 hour sample is stored at 4° C., while theother samples are stored at 37° C. FIG. 5 shows that the longer thecompositions are stored, the less active it becomes. i.e., the lessprotective effect on infection Peptide T has.

EXAMPLE 8 Effects of TFE Concentration and Time and Temperature ofTreatment on DAPTA Aggregation

Effect of TFE Concentration on DAPTA Aggregation.

DAPTA was synthesized by Peninsula Labs, CA (95% pure). The peptide wasdissolved in the indicated concentration of TFE in water and then shakenor agitated for 24 hrs at 37° C. See the Table below. The peptide wassubsequently dried down, and the TFE/water mixture removed under vacuum.Dried DAPTA was reconstituted or resuspended in an aqueous solution,e.g., water, and stored for about 3 days until the activity of thepeptide was assayed for antiviral activity. The studies were done intriplicate using 0.1 nM DAPTA and the results are presented with themean in Table VIII below. Although TFE is removed, residual traces ofTFE within acceptable range for human consumption may remain.

Inhibition of HIV infection was studied by utilizing an infection assayusing GHOST CD4 as previously described by Ruff, M R et al., 2001. CCR5cells are infected with HIV BaL, an R5 tropic isolate. Infection isdetected via viral induction of the hGFP gene (green fluorescentprotein) about 48 hours post-infection. Trays were measured in a platereader to determine fluorescence intensity. All infections wereperformed by adding fresh culture medium containing approximately 1,000infectious units of HIV-1 per well (96-well plates).

TABLE VIII Effect of TFE Concentration on DAPTA Aggregation PercentReduction TFE Concentration (%) in HIV Infection 0 0 ± 2 20 0 ± 3 40 0 ±2 60 20 ± 4  80 100 ± 5  100 98 ± 3 

Effect of Time Shaking in TFE on DAPTA Aggregation.

DAPTA, as above, was dissolved in about 80% TFE/water solution for andshaken for various periods of time at 37 degrees Celcius. See Table IXbelow. Again, the peptide was then evaporated or dried down, andresuspended in an aqueous solution, e.g., water for about three days,and tested for anti-HIV activity, as above.

TABLE IX Effect of Time Shaking in TFE on DAPTA Aggregation PercentReduction Time (hrs) in HIV Infection 12  70 ± 4 24 100 ± 3 48 100 ± 3

Effect of Temperature of DAPTA Dissolved in TFE on Aggregation.

DAPTA, as above, was dissolved in 80% TFE, then shaken for about 24hours at the indicated temperature(s) below in Table X. Again, thepeptide was then evaporated or dried down, and resuspended in an aqueoussolution, e.g., water for about three days, and tested for anti-HIVactivity, as above.

TABLE X Effect of Temperature of DAPTA Dissolved in TFE on AggregationPercent Reduction Temp. ° C. in HIV Infection Room Temp.  70 ± 6 37 100± 5

EXAMPLE 9 Stabilization of DAPTA Using Various Formulations

Human elutriator purified monocytes were differentiated into macrophagesby culture for 7 days (Ruff, M R, et al 2001). HIV-1 (ADA) strain wasadded with or without indicated peptide preparations and infectionproceeded for 2 hrs at 37° C. Virus/peptide mixtures were removed bywashing and cell cultures were maintained for 10 days. Supernatants weresampled and the level of HIV reverse transcriptase was determined as ameasure of viral infection. Cultures were in triplicate and the mean andthe standard deviation are presented.

DAPTA is D-ala¹-peptide T-amide, GMP quality DAPTA was obtained fromBachem. Stability of the peptide formulations was determined byreconstituting peptide powder (0.5 mg/ml) in either 80% trifluoroethanol(TFE)/20% water which was then shaken overnight and taken to dryness ina speed-vac, “TFE Tx”, or alternatively the peptide was not TFE treated.Peptide, TFE treated or not, was then reconstituted (0.5 mg/ml) in wateror the indicated alkylglycoside (1 mg/ml) compositions containing 0.1%EDTA. Samples were then aged by storage at ambient temperature (ca 25°C.) for 14 days prior to antiviral testing. In FIG. 6, A3 denotesdodecyl maltoside and B3 denotes sucrose mono dodecanoate.

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Although the present process has been described with reference tospecific details of certain embodiments thereof in the above examples,it will be understood that modifications and variations are encompassedwithin the spirit and scope of the invention. Accordingly, the inventionis limited only by the following claims.

1. A method for increasing the stability of parathyroid hormone (PTH),fragment PTH 1-34, or fragment PTH 3-34 comprising: admixing theparathyroid hormone, fragment PTH 1-34, or fragment PTH 3-34 and astabilizing agent to form a composition, wherein the stabilizing agentis at least one alkylglycoside surfactant selected from the groupconsisting of dodecyl maltoside, tridecyl maltoside, tetradecylmaltoside, sucrose mono-dodecanoate, sucrose mono-tridecanoate andsucrose mono-tetradecanoate, thereby increasing the stability of theparathyroid hormone, fragment PTH 1-34, or fragment PTH 3-34.
 2. Themethod of claim 1, wherein the composition further comprises a mucosaldelivery-enhancing agent selected from the group consisting of anaggregation inhibitory agent, a charge-modifying agent, a pH controlagent, a degradative enzyme inhibitory agent, a mucolytic or mucusclearing agent, a chitosan, and a ciliostatic agent.
 3. The method ofclaim 2, wherein the composition further comprises benzalkonium chlorideor chloroethanol.
 4. The method of claim 1, wherein the compositionfurther comprises a membrane penetration-enhancing agent selected fromthe group consisting of a surfactant, a bile salt, a phospholipidadditive, a mixed micelle, a liposome, a carrier, an alcohol, anenamine, a nitric oxide donor compound, a long-chain amphipathicmolecule, a small hydrophobic penetration enhancer, a sodium or asalicylic acid derivative, a glycerol ester of acetoacetic acid, acyclodextrin or beta-cyclodextrin derivative, a medium-chain fatty acid,a chelating agent, an amino acid or salt thereof, an N-acetylamino acidor salt thereof, an enzyme degradative to a selected membrane componentand any combination thereof.
 5. The method of claim 1, wherein thecomposition further comprises a modulatory agent of epithelial junctionphysiology.
 6. The method of claim 1, wherein the method furthercomprises lyophilizing the composition.
 7. The method of claim 6,wherein the composition retains greater than 50% biological activityupon reconstitution.
 8. The method of claim 1, wherein the compositionis stable for at least one month when stored at temperatures from about25 to 37 degrees Celsius.
 9. The method of claim 1, wherein thecomposition is stable for at least one year when stored at about 4degrees Celsius.
 10. The method of claim 1, wherein the stability of thecomposition is determined by determining the bioactivity of theparathyroid hormone (PTH), fragment PTH 1-34, or fragment PTH 3-34 in anin vivo or in vitro assay.
 11. The method of claim 1, wherein thecomposition further comprises a bulking agent selected from the groupconsisting of albumin, collagen, alginate, and mannitol.
 12. The methodof claim 6, wherein the composition is reconstituted.
 13. A method forreducing aggregation of a parathyroid hormone (PTH), fragment PTH 1-34,or fragment PTH 3-34 comprising: admixing the parathyroid hormone (PTH),fragment PTH 1-34, or fragment PTH 3-34 and an aggregation reducingagent, wherein the aggregation reducing agent is at least onealkylglycoside surfactant selected from the group consisting of dodecylmaltoside, tridecyl maltoside, tetradecyl maltoside, sucrosemono-dodecanoate, sucrose mono-tridecanoate and sucrosemono-tetradecanoate, thereby reducing aggregation of the parathyroidhormone (PTH), fragment PTH 1-34, or fragment PTH 3-34.
 14. A method forreducing immunogenicity of a parathyroid hormone (PTH), or fragment PTH1-34, or fragment PTH 3-34 upon administration to a vertebrate,comprising: a) admixing a parathyroid hormone (PTH), or fragment PTH1-34, or fragment PTH 3-34 and an immunogenicity reducing agent to forma composition, wherein the immunogenicity reducing agent is at least onealkylglycoside surfactant selected from the group consisting of dodecylmaltoside, tridecyl maltoside, tetradecyl maltoside, sucrosemono-dodecanoate, sucrose mono-tridecanoate and sucrosemono-tetradecanoate; and b) administering the composition to avertebrate thereby reducing immunogenicity upon administration of theparathyroid hormone (PTH), or fragment PTH 1-34, or fragment PTH 3-34.15. A method of manufacturing a non-aggregated aqueous solution of aparathyroid hormone (PTH), or fragment PTH 1-34, or fragment PTH 3-34comprising: admixing the parathyroid hormone (PTH), or fragment PTH1-34, or fragment PTH 3-34 and at least one alkylglycoside surfactant toform an aqueous solution, wherein the surfactant is selected from thegroup consisting of dodecyl maltoside, tridecyl maltoside, tetradecylmaltoside, sucrose mono-dodecanoate, sucrose mono-tridecanoate andsucrose mono-tetradecanoate, thereby manufacturing the non-aggregatedaqueous solution.